FGF Signaling Is Required for Lens Regeneration in

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pcDNA-GPC3真核表达载体的构建及鉴定

pcDNA-GPC3真核表达载体的构建及鉴定李莉;李庆;乔路新;丁渭;吕福东【摘要】目的构建人类GPC3重组真核表达载体.方法以人类肝脏cDNA文库为模板扩增出GPC3基因序列,应用T克隆载体及pcDNA真核表达载体重构,将质粒转化进入HLE人肝癌细胞系,进行荧光免疫方法检测.结果测序结果序列正确,荧光染色转染质粒的HLE细胞可见细胞浆和细胞膜上有GPC3的表达.结论成功构建真核表达载体pcDNA-GPC3,并在真核细胞中可以表达.%Objective To construct the cukaryotic expression vector of the human GPC3 gene. Methods To get the human GPC3 cDNA from human liver cDNA library,and then inserted the fragment of GPC3 into pMD-18 simple T vector. The amplified products were cloned into the cukaryotic expression vector pcDNA 4. 1. The rccombinant piasmid was transfected into HLK cells,and analyzed by immunoflurcseence method. Results The rccombinant plasmid was identified to be right for ORF by restriction enzyme analysis and DNA sequencing. The green fluorescence could be seen in transfected HLE cell membrane and cytoplasm under fluorescence microscope. Conclusion To construct the rccombinant cukaryotic expression vector and express GPC3 protein in HLE cells successfully.【期刊名称】《中国实验诊断学》【年(卷),期】2012(016)001【总页数】3页(P5-7)【关键词】GPC3;基因表达;融合蛋白【作者】李莉;李庆;乔路新;丁渭;吕福东【作者单位】首都医科大学附属北京佑安医院,北京100069;首都医科大学附属北京佑安医院,北京100069;首都医科大学附属北京佑安医院,北京100069;首都医科大学附属北京佑安医院,北京100069;首都医科大学附属北京佑安医院,北京100069【正文语种】中文【中图分类】Q78磷脂酰肌醇蛋白聚糖-3（GPC3）是一种硫酸乙酰肝素糖蛋白（heparansulfate proteoglycan，HSPG），通过磷脂酰肌醇锚定在细胞膜上［1，2］。

J Mol Cell Biol-2011-Ollila-jmcb_mjr016

ReviewThe tumor suppressor kinase LKB 1:lessons from mouse modelsSaara Ollila and Tomi P.Ma¨kela ¨*Institute of Biotechnology,University of Helsinki,Viikki Biocenter,Viikinkaari 9B,FIN-00014,Helsinki,Finland*Correspondence to:Tomi P.Ma¨kela ¨,E-mail:tomi.makela@helsinki.ﬁMutations in the tumor suppressor gene LKB 1are important in hereditary Peutz–Jeghers syndrome,as well as in sporadic cancersincluding lung and cervical cancer.LKB 1is a kinase-activating kinase,and a number of LKB 1-dependent phosphorylation cascades regulate fundamental cellular and organismal processes in at least metabolism,polarity,cytoskeleton organization,and prolifer-ation.Conditional targeting approaches are beginning to demonstrate the relevance and speciﬁcity of these signaling pathways in development and homeostasis of multiple organs.More than one of the pathways also appear to contribute to tumor growth fol-lowing Lkb 1deﬁciencies based on a number of mouse tumor models.Lkb 1-dependent activation of AMPK and subsequent inacti-vation of mammalian target of rapamycin signaling are implicated in several of the models,and other less well characterized pathways are also involved.Conditional targeting studies of Lkb 1also point an important role of LKB 1in epithelial–mesenchymal interactions,signiﬁcantly expanding knowledge on the relevance of LKB 1in human disease.Keywords:LKB 1,tumor suppressor,mouse model,AMPKIntroductionCancer arises as a result of accumulating genetic and epige-netic changes,which compromise the cell’s ability to control its identity and proliferation.Many identiﬁed tumor suppressors play a well-established role in regulation of cell growth and div-ision (e.g.Rb,APC,p 21,PTEN)and genome maintenance (e.g.p 53,BRCA 1-2,ATM,ATR,MLH 1,MSH 2),providing a logical link between the loss of gene product and promotion of carcinogen-esis.An interesting exception is the serine /threonine kinase gene LKB 1(also known as STK 11),which has in recent years taken a prominent position among tumor suppressors.Heterozygous germline mutations in LKB 1predispose to Peutz–Jeghers syndrome (PJS)where patients develop benign polyps in the gastrointestinal (GI)tract and are in high risk of developing malignant tumors in GI tract,breast,and gyneco-logical organs (Giardiello et al .,2000).Importantly,somatic LKB 1mutations are found at least in lung (Ji et al.,2007)and cervical cancer (Wingo et al .,2009).Through phosphoryl-ation of several cellular kinases LKB 1has been implicated in control of cellular and organismal metabolism,cell polarity,and a variety of other functions ranging from proliferation and migration to senescence,apoptosis,DNA damage responseand differentiation (Vaahtomeri and Ma¨kela ¨,2011).Despite these many functions attributed to LKB 1,their speciﬁc contri-butions to the maintenance of tissue homeostasis in vivo and tumor growth are only sketchily appearing with thedevelopment of LKB 1mouse models.This work is important to enable rational treatment strategies to LKB 1-deﬁcient tumors.The LKB 1kinase acts in a trimer with a pseudokinase STRAD and the scaffold protein MO 25to phosphorylate at least 14kinases with conserved activation sites (Katajisto et al.,2007).A well-known substrate of LKB 1is AMPK,which is the master reg-ulator of cellular and organismal metabolism,providing a putative downstream pathway to LKB 1-mediated tumor suppression (Shackelford and Shaw,2009).In mouse studies,AMPK requires LKB 1for activation in vivo in most tissues (Sakamoto et al .,2005;Shaw et al .,2005;Contreras et al .,2008;Hezel et al .,2008).AMPK senses the energy state of cells through monitoring AMP levels as a sensitive readout for ATP.AMPK is activated following exercise,hypoxia,or glucose deprivation,after which it phosphor-ylates multiple targets to increase energy uptake and catabolic processes such as glucose uptake and fatty acid oxidation,and suppress anabolic processes such as lipogenesis and cholesterol synthesis (Hardie et al.,2003).AMPK is the potential candidate to mediate LKB 1’s effects in cell growth via the mammalian target of rapamycin (mTOR)signal-ing (Corradetti et al .,2004;Shaw et al .,2004),which is the pathway monitoring the availability of nutrients in regulation of cell size and protein synthesis as well as proliferation (Zoncu et al.,2011).Increased mTOR signaling is common in cancer (Guertin and Sabatini,2007)and also present in at least some Lkb 1-deﬁcient tumors (Shaw et al.,2004;Ji et al.,2007;Contreras et al.,2008;Hezel et al.,2008;Shackelford et al.,2009).An additional link between LKB 1and mTOR pathway#The Author (2011).Published by Oxford University Press on behalf of Journal ofMolecular Cell Biology ,IBCB,SIBS,CAS.All rights reserved.doi:10.1093/jmcb /mjr 016Journal of Molecular Cell Biology (2011),Vol no.0,1–11|1Journal of Molecular Cell Biology Advance Access published September 15, 2011 at Shihezi University on September 27, 2011 Downloaded frommay lie in regulation of PI 3K-Akt pathway inhibitor PTEN by LKB 1(Mehenni et al .,2005).Loss of cell polarity is commonly noted in cancer,and LKB 1is an important factor for cell polarity in different organisms.In C.elegans ,the orthologs for LKB 1(par-4)and MARK s (par-1)were identiﬁed in a panel of six partitioning (par )mutants which disrupted the polarity of the early embryos (Kemphues et al.,1988).In Drosophila ,Lkb 1is required for proper oocyte polarity (Martin and St Johnston,2003).In mammalian cells,in both 2D and 3D cell culture models and in vivo ,LKB 1is known to regulate polarity (Baas et al .,2004;Partanen et al .,2007;Hezel et al .,2008).Polarity defects are,however,not seen in all Lkb 1-deﬁcient tumors (Contreras et al.,2008,2010).Several of the LKB 1substrates have been reported to mediate the regulation of cell polarity through regulating the cytoskeleton and formation of cell–cell junctions.MARK kinases are implicated in the stability of microtubules by phosphorylating and thereby dissociation microtubule-associated proteins (MAPs),for example the tau protein,from microtubules (Drewes et al .,1997;Stoothoff and Johnson,2005).Neuronal polarity and axon formation are regu-lated by LKB 1at least partially via BRSK kinases (Kishi et al.,2005;Barnes et al.,2007).To what extent LKB 1acts as a polarity protein in mammalian non-neuronal cells still remains to be deter-mined,although at least in both exo-and endocrine pancreas Lkb 1loss leads to polarity defects in vivo (Hezel et al .,2008;Granot et al .,2009).As formation of stress ﬁbers is essential incell contractility,recent studies associate LKB 1with cell motility via NUAK 1and NUAK 2,which have been implicated in regulation of myosin light chain phosphorylation (Vallenius et al.,2010;Zagorska et al.,2010).For detailed information of the molecular signaling pathways of Lkb 1,the reader is recommended recent reviews more focused on that topic (Katajisto et al.,2007;Hezel and Bardeesy,2008;Vaahtomeri and Ma¨kela ¨,2011).Role of Lkb 1in development and tissue homeostasis in miceAlthough LKB 1is a tumor suppressor,inactivation of Lkb 1through homologous recombination or ‘knock-out’(KO)does not always lead to tumors.This is due partly to essential functions of Lkb 1in development and partly demonstrates the tissue-speciﬁcity of Lkb 1functions,where in some cell types biallelic deletion is detrimental to cells or affects speciﬁc functions in metabolism as summarized in Figure 1and discussed below.Role of Lkb 1in embryogenesisGeneration of full KO revealed that Lkb 1is essential for embry-ogenesis;no viable Lkb 12/2embryos were seen after E 11.Analysis of the E 8.5–E 9.5embryos revealed severe developmen-tal defects including impaired neural tube closure and somitogen-esis,mesenchymal tissue cell death,and defective vasculature.The extra-embryonic tissues (yolk sac and placenta)were also deformed.VEGF signaling was highly upregulated in theKOFigure 1Non-tumorigenic phenotypes following Lkb 1targeting in mice.Phenotypes (green)are grouped according to tissue type,cell typeaffected /analyzed (blue),and alleles used for targeting.When appropriate,activator of deletor is indicated in purple.Noted signaling change(s)indicated in red.Alleles as displayed in original publications except for Lkb 1ﬂox 2h /ﬂox 2h hypomorphic Lkb 1(Sakamoto et al,2005).(1)Londesborough et al.,2008;(2)Ohashi et al.,2010;(3)Cao et al.,2010;Tamas et al.,2010;(4)Shorning et al.,2009;(5)Woods et al.,2011;(6)Shaw et al.,2005;(7)Sun et al.,2010a ;(8)Sun et al.,2011;(9)Granot et al.,2009;Fu et al.,2009;(10)Koh et al.,2006;(11)Sakamoto et al.,2005;(12)Sakamoto et al.,2006;Jessen,et al.,2010;(13)Ikeda et al.2009;(14)Gurumurthy et al.,2010;Nakada et al.,2010;(15)Gan et al.,2010;(16)Barnes et al.,2007;(17)Ylikorkala et al.,2001.tam,tamoxifen;b -NF,b -naphtoﬂavone;pIpC,polyinosinic–polycytidylic acid;iv,intravenous.2|Journal of Molecular Cell Biology Ollila and Ma¨kela ¨ at Shihezi University on September 27, 2011 Downloaded fromembryos,possibly relating to the vascular phenotype (Ylikorkala et al .,2001).Embryonic lethality,no embryonic turning,and small somites were also shown in another report of Lkb 1full KO (Jishage et al .,2002).The severe developmental defect was not a result of the abnormal extra-embryonic tissues,since epiblast-speciﬁc conditional inactivation of Lkb 1using Mox 1-Cre resulted in very similar embryonic lethal phenotype to full KO (Londesborough et al .,2008).The important role of Lkb 1in development and maintenance of neurons,mesenchymal cells,and vascularization has been recapitulated in tissue-speciﬁc Lkb 1KOs.Role of Lkb 1in angiogenesisLondesborough et al .(2008)further dissected the role of Lkb 1in endothelia by deleting Lkb 1in vascular endothelial cells using Tie 1-Cre (Figure 1).The mice died at E 12.5and displayed dilated embryonic vessels and pericardial swelling.The vessels were irre-gular and distorted and suffered from inadequate supportive vas-cular smooth muscle cell layer.Since Tgf b signaling was reduced both in Lkb 1-deﬁcient mouse yolk sacs and human umbilical vein endothelial cells (HUVECs)where LKB 1expression was silenced by siRNA,the vascular phenotype was suggested to result from a loss of supporting vascular smooth muscle cells as a conse-quence of attenuated Tgf b signaling from endothelial cells (Londesborough et al .,2008).Another report also described mice lacking Lkb 1in endothelial cells,deleted using Tie 2-Cre driver (Ohashi et al.,2010)(Figure 1).This study repeated the ﬁnding that endothelial Lkb 1is essential for proper embryonic development and no homozygous mutants were born.Analysis of heterozygous Tie 2-Cre;Lkb 1ﬂox /+mice revealed that the mice,including vasculature,seemed phenotypically normal,but displayed reduced revascularization after hind-limb ischemia.Studies in mouse tissues,primary mouse endothelial cells,and HUVECs implemented that the phenotype was mediated via AMPK (Ohashi et al.,2010).In this study,the authors did not address the contribution of Tgf b signaling to the observed phenotype.In the Tie 2-Cre model,Lkb 1–AMPK axis seemed to mediate proangiogenetic signaling as Lkb 1heterozygosity resulted in reduction of revascularization in adult mice (Ohashi et al.,2010).In developing embryo,increased VEGF signaling upon Lkb 1loss would suggest the opposite,antiangiogenic role for Lkb 1(Ylikorkala et al .,2001).Also in the context of PJS polyps where a loss of Lkb 1leads to increased HIF 1a and vasculature,Lkb 1seems to be rather antiangiogenic (Shackelford et al .,2009).However,reduced capillary density was reported in mice where Lkb 1was conditionally deleted from the heart (Ikeda et al .,2009).In 3D culture system where endothelial cells are embedded in Matrigel,both over-expression (Xie et al .,2009)and inhibition (Ohashi et al.,2010)of Lkb 1have been reported to inhibit network formation,suggesting proper expression of LKB 1is essential for angiogenesis.Thus,the precise role of Lkb 1in angiogenesis seems to be dependent on the tissue type and /or the developmental phase,varying from inhibition to promotion.Role of Lkb 1in liverThe ﬁnding that Lkb 1functions upstream of AMPK (Shaw et al .,2004)led to interest to study its effects in liver,where many path-ways of carbohydrate and lipid metabolism,including glycogen-esis,glycogenolysis,gluconeogenesis,lipogenesis,and cholesterol synthesis take place.Tail-vain injection of Adeno-Cre to mice carrying conditional Lkb 1allele led to hepatocyte-speciﬁc Lkb 1deletion since Adeno-Cre has high tropism for hepatocytes (Shaw et al .,2005)(Figure 1).Lkb 1loss resulted in nearly complete abolishment of AMPK activation in liver,and the glucose metabolism of the mice was impaired demonstrated by elevated blood glucose.CRTC 2phosphorylation was reduced in the livers of the mice,leading to elevated CREB-mediated transcription,including expression of PGC 1a and other gluconeogenetic genes.Also lipogenetic genes were over-expressed.Metformin,the diabetes drug which reduces blood glucose levels via AMPK pathway (Zhou et al .,2001),did not lower blood glucose in the liver-speciﬁc Lkb 1KO mice,demonstrating that AMPK activity induced by Lkb 1in liver is required for the effects of metformin in vivo .In another report of liver-speciﬁc Lkb 1knockout using Alb-Cre driver,Woods et al.(2011)reported defective bile ducts in liver,leading to accumulation of bile in liver and serum (Figure 1).Bile salt export pump was not located in canalicular membrane of the bile canaliculi,indicating possible defects in cell polarity.The mice also suffered from cholestasis (Woods et al.,2011).These reports of liver-targeted deletions of Lkb 1demonstrate the critical requirement of Lkb 1in glucose,lipid,bile,and cholesterol metab-olism.Furthermore,they show that in liver,Lkb 1is the main acti-vator of AMPK,and its activity is required for the AMPK-mediated suppression of lipogenesis and gluconeogenesis to take place.Role of Lkb 1in muscleMuscles are highly energy-consuming tissues whose glucose homeostasis needs to be regulated both in response to insulin after blood sugar increase,and to exercise-mediated deﬁciency of glucose storage.Sakamoto et al.(2005)provided the ﬁrst genetic evidence that Lkb 1is required for AMPK activation in vivo in skeletal muscle.They generated conditional Lkb 1mice in which cDNA of Lkb 1exons 5–7fused with neomycin resistance cassette,surrounded by loxP sites,was inserted between exons 4and 8in the genomic Lkb 1locus.The resulting mice were hypomorphic and expressed only 10%–20%of normal levels of Lkb 1in the absence of Cre -mediated ing MCK-Cre driver to create muscle-speciﬁc Lkb 1KO,they found that AMPK a 2(one of the two alternative catalytic subunits of AMPK)activation either by the AMP analog AICAR,muscle con-traction or phenformin,a similar blood glucose lowering drug to metformin,was lost and AMPK a 1activation greatly reduced.Upon contraction,glucose transport to muscle cells was abol-ished (Sakamoto et al.,2005).In another study using the same muscle-speciﬁc MCK-Cre with another (non-hypomorphic)con-ditional Lkb 1line,effects of Lkb 1loss in muscle to levels of blood glucose were investigated (Koh et al.,2006)(Figure 1).Interestingly,glucose metabolism seemed to be enhanced in these mice,demonstrated by reduced fasting blood glucose and blood insulin concentrations,improved glucose tolerance,and increased muscle glucose uptake.This phenotype,indicating that Lkb 1in muscle functions as a negative regulator of glucose metabolism,was suggested to be resulting from improved muscle glucose uptake,mediated by increased phosphorylation of Akt and reduced the gene expression of the Akt inhibitor TRB 3.Lkb 1loss abolished the activity of AMPK a 2,but notLessons from LKB 1mouse modelsJournal of Molecular Cell Biology |3at Shihezi University on September 27, 2011 Downloaded fromAMPK a 1in muscle cells.Also MARK 4,but not MARK 2/3activitywas signiﬁcantly reduced.Based on this study,the metabolic effects mediated by Lkb 1in muscle seem to oppose those of the liver,at least in terms of blood glucose levels (Koh et al.,2006).Recently,the Lkb 1substrate NUAK 2was proposed to be a mediator of contraction-stimulated glucose transport by skel-etal muscle (Koh et al.,2010).Also cardiac muscle lacking Lkb 1has been investigated.Sakamoto et al.(2006)studied the effect of Lkb 1deﬁciency in heart using the MCK-Cre driver,which deletes Lkb 1in both skel-etal and cardiac myocytes and found that Lkb 1inactivation did not lead to overt cardiac dysfunction,although the weight of the heart was reduced and the atria enlarged;however,the study revealed that cardiac Lkb 1is required for activation of AMPK a 2both in basal conditions and in response to ischemia (Figure 1).Also Jessen et al.(2010)used the MCK-Cre driver but the Lkb 1allele was not hypomorphic as in the Sakamoto et al.(2006)study.They showed that ablation of Lkb 1in heart leads to impaired cardiac function both in basic conditions and post-ischemia and suggested that failure to downregulate mTOR sig-naling by AMPK a 2activation underlined the phenotypes.Ikeda et al.(2009)used a -MHC-Cre to delete Lkb 1speciﬁcally from the heart,and a more severe phenotype was observed:the mice displayed hypertrophy and impaired function of the heart,reduction of cardiac capillary density,and increased ﬁbrosis and collagen content and died by 6months of age.The differ-ences between these phenotypes may reﬂect differences in the timing of Cre activity,speciﬁcity of the Cre recombination,and /or the conditional Lkb 1allele used.However,it seems clear that Lkb 1is needed for the normal function of heart both in basal and ischemic conditions.Role of Lkb 1in pancreasPancreatic b -cells secrete insulin and are thus important mediators of whole-body glucose metabolism.As Lkb 1–AMPK axis is important in regulation of liver metabolism and muscle glucose homeostasis,it is of interest to study whether Lkb 1has an effect on the insulin release.Granot et al.(2009)used the Pdx 1-CreER driver to delete pancreatic Lkb 1in 6-week-old mice by tamoxifen injection (Figure 1).In response to glucose injection,the mutant mice secreted more insulin than control mice,which carried the conditional Lkb 1allele but were not subjected to tamoxifen injection.Deletion of Lkb 1led to increased size of b -cells together with disrupted polarity.Increased mTOR signal-ing seemed to mediate the cell size increase,while the polarity defect took place at least partially through MARK 2.Increased insulin secretion was partially dependent on AMPK (Granot et al .,2009).Fu et al .(2009)used the same Pdx 1-CreER system to delete Lkb 1in adult b -cells and also found that the mice showed improved glucose tolerance,b -cells mass had increased,and mTOR pathway was activated (Figure 1).These results place Lkb 1as an important regulator of pancreatic b -cell size,polarity,and function,further highlighting its essence in regulation of organismal metabolism.Sun et al.(2010a)investigated pancreatic b -cells with the Rip 2-Cre driver,which activates Cre -mediated recombination in pancreatic b -cells and some hypothalamic neurons,and found that the mice displayed diminished food intake and weight gain,enhanced insulin secretion,and improved glucose tolerance (Figure 1).Also here,mTOR pathway was activated.However,the study by the same group where both AMPK a subunits were deleted in b -cells using the same Rip 2-Cre showed decreased insulin secretion (Sun et al.,2010b ).This suggests that Lkb 1loss regulates mTOR signaling in b -cells partially independent of AMPK,or that the hypothalamic Lkb 1and AMPK have different functions,impacting the feeding behavior and hormonal balance.Role of Lkb 1in immune systemThree recent studies elegantly demonstrated that Lkb 1regu-lates the quiescence and maintenance of hematopoietic stem cells (HSCs)using conditional Lkb 1alleles with Mx 1-Cre followed by injections of polyinosinic–polycytidylic acid (pIpC),or Rosa 26-CreERt 2followed by tamoxifen injections (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010)(Figure 1).Both approaches resulted in a similar phenotype:increased pro-liferation followed soon by decline in HSC number,resulting in loss of all immune cell types (pancytopenia)and death.Transplantation experiments demonstrated that Lkb 1-deﬁcient HSCs were not able to reconstitute the bone marrow of irradiated wild-type (wt)mice,nor were they able to compete with wt donor cells,demonstrating that the effect was cell-autonomous;mito-chondrial defects and decreased ATP levels,as well as altered long-chain fatty acid and nucleotide metabolite levels suggested metabolic defects to underlie the phenotypes noted (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010).Interestingly,only minor similarities in mitochondrial phenotypes were found when mice defective for both AMPK a subunits were compared with Lkb 1KO mice (Nakada et al.,2010),implicating other Lkb 1substrates in these phenotypes.Consistent with this,rapamycin or AMPK activators AICAR and A 769662did not rescue the phenotype in any of the studies.Immune cell apopto-sis was increased,and Lkb 1-deﬁcient HSCs also demonstrated increased autophagy in bone marrow,and inhibiting this further decreased immune cell survival (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010).This would suggest that Lkb 1in this context is suppressing autophagy,whereas previously it has been reported to activate it following elevation of reactive oxygen species (Alexander et al.,2010).Yet another phenotype potentially decreasing HSC viability was the noted increase in supernumerary centrosomes,aberrant mitotic spindles,and aneuploidy (Nakada et al.,2010),which could be due to compro-mised BRSK 2activity (Alvarado-Kristensson et al.,2009).Recently,two groups generated mice where Lkb 1expression is speciﬁcally abolished in the T cell progenitors using the proximal p 56lck-Cre promoter.The studies demonstrate severe deﬁciency in survival and proliferation of T cell progenitors and mature T cells in the absence of Lkb 1(Cao et al.,2010;Tamas et al.,2010)(Figure 1).Also the survival of isolated peripheral T cells in vitro was dependent on Lkb 1(Tamas et al.,2010).Transfection of thymo-cytes with constitutively active AMPK a 2partially rescued the thy-mocytes from cell death,indicating that thymocyte survival is mediated at least via AMPK pathway (Cao et al.,2010).Thus,the common hematopoietic cell precursors and T cell precursors seem to have different requirement for AMPK signaling,although cell sur-vival is defective in both cell types in the absence of Lkb 1.The studies in hematopoietic cells have revealed an interesting aspect4|Journal of Molecular Cell Biology Ollila and Ma¨kela ¨ at Shihezi University on September 27, 2011 Downloaded fromof Lkb 1biology:although being a tumor suppressor in some tissues,in others Lkb 1is required for survival.Role of Lkb 1in nervous systemLkb 1KO embryos exhibit severe deﬁciencies in development of neuronal tissues (Ylikorkala et al .,2001).Since LKB 1orthologs in nematodes and fruit ﬂies have been identiﬁed through their indis-pensable role in establishing polarity (Kemphues et al .,1988;Martin and St Johnston,2003)and LKB 1regulates polarity also in some mammalian cells (Baas et al .,2004;Partanen et al .,2007),it was of interest to generate models which would reveal the in vivo relevance of Lkb 1in establishing the axon-dendrite polarity in neuronal cells.Barnes et al.(2007)deleted Lkb 1in cer-ebral cortex of developing mice using Emx-Cre driver and showed that Lkb 1and its substrates BRSK 1and BRSK 2are required for axon speciﬁcation in the studied neurons.This ﬁnding conﬁrmed the previously described role of BRSK kinases in neuronal polar-ization (Kishi et al .,2005),and placed Lkb 1as the upstream kinase required for the polarization to take place.Lkb 1-activated BRSKs were shown to modify the cytoskeleton by phosphorylating MAPs (Barnes et al.,2007).Studies in rat hip-pocampal neurons in vitro and developing rat cortical neurons in vivo agreed with the ﬁnding that Lkb 1is essential in establishing neuronal polarity;there,lack of either Lkb 1or STRAD prevented axon differentiation (Shelly et al.,2007).Interestingly,over-expression of Lkb 1and STRAD resulted in formation of multiple axons.PKA-mediated phosphorylation of Lkb 1Ser 431was shown to be required for the axon speciﬁcation (Barnes et al.,2007;Shelly et al.,2007).Thus,Lkb 1activity is modulated by upstream factors in a tissue-and context-speciﬁc manner.Not only axon speciﬁcation but also maintenance seems to be regulated via Lkb 1in some systems.Sun et al.(2011)reported,using the pancreatic and hypothalamic Rip 2-Cre ,that the mice developed hind-limb paralysis due to axon degeneration in thor-acic spinal cord neurons at about 7–8weeks of age (Figure 1).The Rip 2-Cre was found to be active also in spinal cord,especially in the thoracic segments.Deleting both AMPK a subunits did not result in axon degeneration or paralysis,and the authors specu-lated that in the absence of Lkb 1,the neuronal polarization and axon degeneration defects might be mediated by BRSK kinase pathways (Sun et al.,2011).PJS and its mouse modelsLKB 1was linked to human disease when its mutations were found to be causative for PJS (Hemminki et al .,1998;Jenne et al .,1998).A major manifestation in PJS is the appearance of large occluding hamartomatous polyps in the GI tract (Giardiello and Trimbath,2006).Mice carrying one inactivated allele of Lkb 1(Lkb 1+/2)recapitulate PJS by developing hamartomatous GI polyps which are indistinguishable from PJS patient polyps (Bardeesy et al .,2002;Jishage et al .,2002;Miyoshi et al .,2002;Rossi et al .,2002)(Figure 2),although in mice polyps appear more in the stomach and less in the small intestine.Polyps appear at 4–6months (Udd et al.,2010),and lead to lethality at an average age of 11months due primarily to obstructions.Biallelic loss of wt Lkb 1is not a prerequisite for polyp formation,indicating that Lkb 1is a haploinsufﬁcient tumor suppressor at least in the context of PJS polyps (Jishage et al .,2002;Miyoshiet al .,2002;Rossi et al .,2002).Strong up-regulation of COX 2has been identiﬁed in the mouse and also PJS patient polyps (Rossi et al .,2002),and COX 2inhibitors have been shown to be efﬁcient suppressors of PJS polyps (Udd et al .,2004).PJS is associated with elevated risk of cancer,especially in the GI tract,and also in breast,pancreas and gynecological cancers (Giardiello and Trimbath,2006;Hearle et al .,2006;Mehenni et al .,2006).Lkb 1+/2mice in turn have been reported to have increased frequency of cancer in liver (Nakau et al .,2002),bones (Robinson et al .,2008),and endometrium (Contreras et al .,2008)(Figure 2).Polyposis in Lkb 1+/2mice is accelerated in a p 53-deﬁcient background (our unpublished data)(Wei et al .,2005;Takeda et al .,2006)(Figure 2),and p 53mutations are detected in the GI cancers of PJS patients (Miyaki et al .,2000).Despite these observations,progression of the benign hamarto-matous polyps to dysplasia or carcinoma is not clearly estab-lished possibly due to the rapid growth of the hamartomatous polyps leading to GI occlusions.As haploinsufﬁciency of Lkb 1is sufﬁcient for polyp initiation (Jishage et al .,2002;Miyoshi et al .,2002;Rossi et al .,2002)though biallelic loss has been noted (Bardeesy et al .,2002),loss of the remaining allele of Lkb 1may represent a progression step,although it has also been suggested that the loss of Lkb 1is associated with the resist-ance to progression in this context (Bardeesy et al.,2002).Mesenchymal Lkb 1loss leads to PJS-type polyposis in mice PJS polyps are classiﬁed as hamartomatous polyps thought to contain all the cell types of the surrounding tissue.However,it was recently noted that epithelial differentiation is disrupted in gastric and small intestinal polyps in Lkb 1+/2mice (Udd et al.,2010),but the model did not enable distinguishing whether this was a cell autonomous function of Lkb 1in epithelial cells.Biallelic disruption of Lkb 1in GI epithelia lead to imbalanced differentiation and positioning of epithelial cells (Shorning et al .,2009)(Figure 1),but was not reported to be associated with tumorigenesis.Polyps in both PJS patients and Lkb 1+/2mice harbor a large component of smooth muscle tissue.Remarkably,in a mouse model,where Lkb 1deﬁciency was restricted to the smooth muscle lineage by using a tamoxifen-inducible SM 22-CreERt 2line,PJS type polyps appeared in stomachs of the mice with the hetero-and homozygous Lkb 1mutants (Katajisto et al .,2008)(Figure 2).The polyps appeared later than those in the Lkb 1+/2mice,suggesting either that tamoxifen-induced Lkb 1loss at 6weeks of age delayed the poly-posis,or that mesenchymal loss of Lkb 1signaling is sufﬁcient to drive hyperproliferation of epithelial tissue,but that coexisting epithelial mutations accelerate the process.This interesting aspect of Lkb 1signaling in tissue interactions is discussed later.Other Lkb 1tumor mouse modelsInactivating LKB 1mutations are associated with the develop-ment of cancer in several tissues.Various strategies of targeted inactivation of Lkb 1in mice,sometimes in combination of other tumorigenic mutations,have led to the development of various types and grades of tumors in multiple tissues,sometimes mod-eling human cancers in very useful ways as discussed below and summarized in Figure 2.Lessons from LKB 1mouse modelsJournal of Molecular Cell Biology |5at Shihezi University on September 27, 2011 Downloaded from。

Links between Tumor Suppressors p53 Is Required for TGFβ Gene Responses by Cooperating with Smads

Cell,Vol.113,301–314,May 2,2003,Copyright 2003by Cell PressLinks between Tumor Suppressors:p53Is Required for TGF-␤Gene Responses by Cooperating with Smadsphosphorylation and activation of the Smad family of signal transducers.Two different Smad signaling branches have been described:TGF-␤-like signals,including TGF-Michelangelo Cordenonsi,1Sirio Dupont,1Silvia Maretto,1Alessandra Insinga,2Carol Imbriano,3and Stefano Piccolo 1,*␤s,Activin,and Nodal,are transduced by Smad2or 1Department of Histology,Microbiology,and Smad3,whereas BMPs are transduced by Smad1(Mas-Medical Biotechnologiessague ´,2000).Section of Histology and Embryology Once activated,the Smads translocate into the nu-University of Padua cleus where they control gene expression in association viale Colombo 3with Smad4and partner transcriptional regulators (Mas-35121Padua sague ´,2000).How the Smads recognize and properly Italy activate a specific promoter is not fully understood.In 2Department of Experimental Oncology part,specificity depends on the differential expression European Institute of Oncology of distinct Smad partners in distinct cell types;however,via Ripamonti 435several inputs can profoundly modify both the percep-20141Milan tion of the TGF-␤signal and its biological output (Mas-Italy sague ´,2000).Understanding how this transcriptional 3Department of Biologyplasticity is attained is central for embryonic develop-University of Modena and Reggio Emilia ment and cancer.For example,most carcinomas have via Campi 213selectively lost the growth arrest response and gained 41100Modena metastatic abilities in response to TGF-␤(Wakefield and ItalyRoberts,2002).Importantly,this change can occur with-out acquiring genetic defects in known components of the TGF-␤pathway,indicating that alterations in other Summaryregulatory molecules can have a profound influence on the cellular responsivenss to TGF-␤.Some of these reg-The p53tumor suppressor belongs to a family of pro-ulators appear to act in parallel to the Smad signal trans-teins that sense multiple cellular inputs to regulate cell duction cascade,namely converging at the level of tar-proliferation,apoptosis,and differentiation.Whether get gene expression (Lehmann et al.,2000).and how these functions of p53intersect with the ac-To further understand the molecular mechanisms that tivity of extracellular growth factors is not understood.control of TGF-␤gene responses,we performed an un-Here,we report that key cellular responses to TGF-␤biased screen for TGF-␤modulators.Here,we report the signals rely on p53family members.During Xenopus unexpected identification of p53as an in vivo relevant embryonic development,p53promotes the activation partner of Smad2in the activation of multiple TGF-␤of multiple TGF-␤target genes.Moreover,mesoderm target genes.p53is a key tumor suppressor in mammals differentiation is inhibited in p53-depleted embryos.In as it is mutated,or inactive,in the majority of human mammalian cells,the full transcriptional activation of tumors (Vogelstein et al.,2000).p53belongs to a family the CDK inhibitor p21WAF1by TGF-␤requires p53.p53-of proteins,including p63and p73,that have evolved deficient cells display an impaired cytostatic response pleiotropic—and perhaps overlapping—cellular func-to TGF-␤signals.Smad and p53protein complexes tions (Yang and McKeon,2000).converge on separate cis binding elements on a target We find that several TGF-␤target genes are under promoter and synergistically activate TGF-␤induced joint control of p53and Smads.p53binds to Smads in transcription.p53can physically interact in vivo with vivo and strongly cooperates transcriptionally with the Smad2in a TGF-␤-dependent fashion.The results un-activated Smad complex.In Xenopus embryos and hu-veil a previously unrecognized link between two pri-man cells,TGF-␤requires the assistance of p53to medi-mary tumor suppressor pathways in vertebrates.ate the activation of key TGF-␤target genes and to carry out some of its biological ing the Mix.2Introductionpromoter as a paradigm,we find that p53adjusts TGF-␤-induced transcription by interacting directly with a Members of the TGF-␤growth factor family are promi-cognate binding site on promoter DNA.However,differ-nent signals regulating cellular fates in a variety of physi-ent from other Smad partners,this p53binding element ological contexts,from embryonic development to adult is located in a separate position from the Activin/TGF-␤tissue homeostasis (Massague ´,2000).The loss of this responsive element.We argue that these findings unveil control leads to aberrant cell behaviors contributing to a convergence of the p53and the Smad signaling net-the development of cancer and inborn defects (Wake-works to regulate development and tissue homeostasis.field and Roberts,2002).In recent years,tremendous progress has been made Resultsin the elucidation of how cells sense and transduce TGF-␤signals.TGF-␤ligands bind to cognate serine/Cloning of an Alternatively Spliced Isoform of p53threonine kinase receptors leading,intracellularly,to(p53AS)in a Screen for Activators of TGF-␤Signaling To identify molecules that modulate TGF-␤/Activin/Nodal signaling during development,we performed an*Correspondence:piccolo@civ.bio.unipd.itCell302unbiased functional screen for genes whose expression nally modified p53isoform has not been described in promoted the differentiation of embryonic cells into en-human or frog cells that express regular p53(p53R) doderm and mesoderm,as this is the hallmark of TGF-␤(Wolkowicz et al.,1995).Xenopus and mammalian p53 signaling in early vertebrate embryos(Whitman,2001).proteins share similar functional properties and regula-We generated a mouse gastrula(embryonic day[E]6.5)tory mechanisms(Cox et al.,1994).To assay for func-cDNA library constructed in an RNA expression plasmid.tional conservation,we compared the ability of mouse Synthetic mRNA was prepared from pools of100bacte-p53AS,human p53R,and Xenopus p53(Xp53)to induce rial colonies and injected into the animal hemispheremesoendoderm differentiation in animal cap assays. of2-cell Xenopus embryos.At the blastula stage,the Figure1C shows that the inducing activities of mp53AS ectoderm was explanted and cultivated until siblingsand Xp53are similar.hp53R can also stimulate expres-reached the gastrula stage.The injected animal caps sion of the same marker genes but at5-to10-fold lower were then assayed by RT-PCR to identify pools able toefficiency than mp53AS,perhaps revealing a partial in-activate the expression of Mixer(endoderm)and Xbra hibitory role for p53C terminus in these activation pro-(mesoderm).Of five positive pools,two of the activecesses.Of note,injection of higher doses(above400 cDNAs isolated after sib selection corresponded to pg for all mRNAs)was detrimental for survival(data not Smad2(Baker and Harland,1996)and,unexpectedly,shown).three corresponded to p53AS,a natural variant of p53p53may stimulate TGF-␤gene responses acting in generated by alternative splicing at the C terminus(Wol-partnership with endogenous Smads or,alternatively, kowicz et al.,1995).p53AS shares with commonly operating in an independent pathway.To discriminate spliced p53(p53R)the N-terminal transactivation do-between these two possibilities,we tested whether a main,the central DNA binding and oligomerization do-blockade of Smad function had an effect on p53-medi-mains,but lacks the most C-terminal26amino acids ofated gene expression.As shown in Figure1D,coinjec-p53R.tion of p53AS and dominant-negative Smad2(Candia A wealth of data indicates that the TGF-␤and p53et al.,1997)mRNAs downregulated all the TGF-␤-like signaling networks operate independently as powerful inductions triggered by p53AS.We conclude that ex-tumor suppressors in mammalian cells;yet,the cloningpression of p53activates the transcription of TGF-␤of a p53isoform in a TGF-␤screen unveiled the possibil-target genes in a Smad-dependent fashion.ity of a previously unrecognized partnership betweenTo explore the possibility that p53and Smad may these two types of molecules.We initially addressed jointly control the TGF-␤output,we tested whether rai-this issue by characterizing the p53AS effects in moresing the levels of p53may correspond to an enhanced detail.Different doses of p53AS mRNA were injected in responsiveness to TGF-␤.Figure1E shows that in animal the animal pole of each blastomere at the2-cell stagecaps explants,coinjection of suboptimal levels of activin and tested for the induction of several tissue-specific and p53AS mRNAs cooperated in the induction of endo-markers in animal cap cells explanted from these em-dermal and mesodermal markers,whereas each compo-bryos(Figure1A).At lower doses of injected mRNA,nent alone was weak or inactive.In contrast,the BMP4 p53AS induced first mesodermal(Xbra,Eomes)and thentarget Vent-1was neither induced by p53AS alone nor mesendodermal(VegT,Mix.2)genes;at a higher con-in combination with BMP4(see Supplemental Figure centration,mainly endodermal markers were turned onS1A online at /cgi/content/full/113/ (Sox17␤,Mixer,Xnr6)(Figure1A,lanes1–5).This pattern3/301/DC1).of gene expression is typical of the ectopic activationWe further tested whether p53-mediated effects are of TGF-␤/Activin/Nodal/Smad2signaling in animal caps direct by assaying the biological activities of p53and (Figure1A,lanes6and7)(Harland and Gerhart,1997).Smad2in the presence of cycloheximide,a protein syn-However,other genes activated by Activin/Smad2such thesis inhibitor.Transcription of Xbra,Mix.2,and Eomes as goosecoid,XWnt8,and Xnr-1were not induced inis initiated as immediate response to Activin/Smad2 p53AS-injected cells(Figure1A,bottom).This suggests stimulation in Xenopus animal caps(Harland and Ger-that p53AS specifically activates a subset of TGF-␤tar-hart,1997)and,as shown in Supplemental Figure S1B, get genes.p53directly promotes transcription of the same genes We tested the biological activity of p53mRNA in thein the absence of de novo protein synthesis.In keeping context of the whole embryo.When microinjected into with this notion,injection of p53alleles bearing inactivat-a single ventral blastomere at the4-cell stage,p53ASing mutations in the DNA binding(R273H)or transactiva-mRNA induced the formation of ectopic trunk-tail struc-tion domain(22-23)failed to induce any mesoendoder-tures(nϭ155,77%),phenocopying the biological ef-mal marker(Figure1F).This suggests that p53relies fects triggered by low doses of Smad2mRNA(Figure1B)entirely on its properties as sequence-specific transcrip-(Baker and Harland,1996).Histological analysis showedtion factor in these inductive events.that these secondary structures contained muscle,neu-p53is biochemically a latent transcriptional regulator ral tissue,and in several cases an ectopic gut,but allthat becomes active in response to a variety of stimuli lacked notochord(data not shown).(Vogelstein et al.,2000).Little is known on the activation We conclude from these experiments that ectopicstatus of Xp53in early embryos.This can be visualized expression of p53stimulates multiple gene responses by monitoring p53-dependent transcription.To this end, and long-term phenotypic effects typically mediated bywe injected at the1-cell stage a luciferase reporter for activation of the TGF-␤signaling cascade in embryonic p53signaling whose transcription is driven by multimer-cells(Harland and Gerhart,1997).ized p53binding elements(PG13)(Kern et al.,1992).We Alternative spliced p53AS represents up to30%of compared PG13transcription with MG13,in which the total p53in rodent cells but,curiously,a similar C termi-p53binding elements are disrupted.Intriguingly,weConvergence of p53and Smads303Figure1.p53Promotes Mesoderm and En-doderm Formation in Xenopus Embryos(A)RT-PCR for mesoderm(Mes)and endo-derm(Endo)markers activated in animal capsexpressing the indicated ne1:lacZ mRNA(200pg).Lanes2–5:p53AS mRNA(3,10,30,150pg,respectively).Lane6:ac-tivin mRNA(30pg).Lane7:Smad2mRNA(200pg).Lane8:eFGF(50pg).Lanes9and10:whole embryo total RNA without(ϪRT)and with reverse transcriptase.(B)Injection of p53AS mRNA(50pg;nϭ155,77%)mimicks the long-term effects of Smad2mRNA overexpression(100pg;nϭ12,100%).Side view of stage28embryos.Dot-ted lines indicate the induced secondarytrunk-tail structures.(C)RT-PCR of animal caps expressing thefollowing mRNAs:hp53R(20pg and200pg),mp53AS(20pg),and Xp53(30pg).(D)TGF-␤-like activities promoted by p53ASmRNA(50pg)are inhibited by dominant-neg-ative Smad2(DNS2:1ng)mRNA.(E)Lane2:inductions by activin mRNA at ahigh dose(35pg)but lower amounts of p53ASor activin mRNA(1pg,each)were effectiveonly in combination(Lanes3–5).Lanes6–8,synergism of p53AS with activin,both usedat5pg of mRNA.(F)RT-PCR on animal caps expressing theindicated mRNAs(200pg each).Lane2:hp53(WT).Lane3:p53L22E-W23S bears an inac-tive transactivation ne4:p53R273H is unable to bind DNA.(G)Detection of p53transcriptional activity inearly embryos.PG13-lux and MG13-lux(40pg per embryo)were injected at the2-cellstage in each blastomere and luciferase ac-tivity was measured on extracts from gastru-lae(nϭ40each).found that Xenopus embryos have considerable endog-protein levels for␤-catenin(Figure2A)or actin(data notshown).enous p53transcriptional activity(Figure1G).We examined the effect of p53knockdown on mes-oendoderm differentiation mediated by Activin protein p53Is Required for TGF-␤/Activin/Nodal-Mediated in animal caps(Figure2B).p53MO and control MO were Gene Responses in Xenopus Embryos injected into the animal hemispheres at the2-cell stage, We sought to establish to what extent p53is required animal caps were removed at blastula and treated in for TGF-␤/Activin/Nodal signaling in vivo.To this end,Activin-containing medium(50pM).As shown in Figure we reduced the endogenous p53protein level with an2B,Activin-mediated inductions of mesodermal(Xbra anti-p53morpholino oligonucleotide(p53MO)covering and Eomes)or endodermal(Mix.2and Mixer)markers the initial codons of Xp53mRNA.As specificity control,were inhibited in p53-depleted caps(Figure2B,com-we used a mutant morpholino oligonucleotide(control pare lanes3and4).Three evidences indicate that this MO).p53MO specifically blocked Xp53mRNA transla-interference is specific.First,the control MO has no tion initiation in vivo,leading to effective knockdown of effect on Activin signaling.Second,normal Activin-the endogenous levels of p53protein,without affectingmediated gene responses can be restored by coinjec-Cell304Figure2.In Vivo Requirement of p53in TGF-␤Gene Responses(A)p53morpholino oligonucleotides(MO,20ng)specifically knockdown the translation ofboth endogenous(left)and overexpressed(right)Xp53mRNA(100pg)in animal halvesexplanted at gastrula stage.␤-catenin proteinserves as specificity control.(B)Animal caps,injected with the indicatedMO,were explanted and treated with Activinprotein(50pM).Lane4:p53MO selectivelyblocks Activin inductions of Mixer,Mix.2,Eomes,and Xbra,but not of goosecoid(Gsc).Lane5:mp53AS mRNA injection rescues Ac-tivin inductions in p53MO injected animalcaps.(C)Whole-mount in situ hybridizations oncontrol-and p53morphant embryos showingsevere phenotypes.Control MO or p53MO(10ng each),together with lacZ mRNA(200pg),were injected at the4-cell stage in a sin-gle blastomere.Embryos shown in the upperpanels were collected at gastrula,stained for␤-gal activity(red staining),and processedfor in situ hybridization.Embryos shown inthe lower panels were radially injected(20ngper embryo)and collected at stage13.Notethat upon p53knockdown,staining forChordin,Xbra,and XmyoD,but not for Vent-1,is reduced.(D and E)Phenotypes of p53-depleted em-bryos.Embryos were injected as in(C)andcultured until siblings reached the tailbudstage.(F–I)Embryos were injected on the right sideat the2-cell stage with15ng of control MO(F)or p53MO either alone(G)or in combina-tion with mp53AS mRNA([H],30pg;[I],100pg).Embryos were collected at stage26andprocessed for in situ hybridization with theXmyoD probe.tion of mouse p53AS,whose translation is not antago-of the pan-mesodermal marker Xbra in early gastrulae;incontrast,expression of Vent-1was not affected(see nized by the p53MO(Figure2B,lane5).Third,Activin-mediated induction of goosecoid is p53independent closer images in Supplemental Figure S2online at http:// (Figure2B,lane4).Collectively,these findings indicate/cgi/content/full/113/3/301/DC1).In a critical role of p53for the activation of a subset of embryos injected in all blastomeres with p53MO,the TGF-␤/Activin targets.development of dorsal mesoderm derivatives was as-We next asked whether p53function was also re-sayed molecularly using probes for Chordin and XmyoD,whose expression at the neurula stage marks axial quired in the whole embryo.Several findings have dem-onstrated that Nodals and Derriere,a group of TGF-mesoderm and prospective skeletal muscle,respec-␤-related ligands,induce and pattern the mesoderm intively.As shown in Figure2C,p53MO,but not control vertebrates(Whitman,2001;Sun et al.,1999).If p53MO,severely downregulated Chordin and XmyoD ex-plays a role in endogenous TGF-␤signaling,p53knock-pression.In monolateral injection experiments(right down should attenuate these inductions in embryos.We side in Figures2F–2I),coinjection of p53MO with in-creasing levels of p53AS mRNA rescues this phenotype. injected p53MO in a single dorsal or ventral blastomereat the4-cell stage and assayed the resulting phenotype At the tailbud stage,major developmental changes oc-using molecular markers by in situ hybridization.Ascurred in p53-depleted embryos compared to control shown in Figure2C,p53MO,but not control MO,attenu-MO injected embryos(Figures2D and2E).Part of the ated the expression of the organizer marker Chordin andp53morphants(23%)failed to gastrulate properly dueConvergence of p53and Smads305to defective blastopore formation.Among the p53mor-activity,although a weaker,p53-independent p21WAF1 phants that survived after gastrulation and neurulation,induction can be observed after hours of Activin sig-several(nϭ76,54%)lacked tail structures and devel-naling.oped a reduced trunk,indicative of defective mesodermOf note,activation of p21WAF1by TGF-␤has been formation.These phenotypes recapitulate aspects of shown to be p53independent in some tumorigenic orimmortalized epithelial cell types(Datto et al.,1995). embryonic TGF-␤deficiencies,such as embryos in-jected with low doses of Cerberus-short,a secreted This indicates,perhaps not surprisingly,that different antagonist of Nodal,or with an inhibitory construct forgenetic programs may be at work in distinct cells(Mas-derriere,both leading to defective trunk development sague´,2000);additionally,it also leaves open the possi-(Piccolo et al.,1999;Sun et al.,1999).bility that p63of p73may be able to compensate for Together,these loss-of-function experiments suggest p53loss-of-function in some contexts.HaCaT cells are that depletion of p53leads to impaired edogenous TGF-a point in case,as this cell line of immortalized keratino-␤/Activin/Nodal gene responses resulting in defective cytes is p53mutant,but highly TGF-␤responsive(Dattoet al.,1995).However,these cells express high levels embryonic development.of p63(Hall et al.,2000),whose reduction by anti-p63siRNA led to a concomitant reduction in of p21WAF1induc-p53Is Required for Full TGF-␤Gene Responsesibility by TGF-␤1(see Supplemental Figure S3online at and TGF-␤-Mediated Growth Arrest/cgi/content/full/113/3/301/DC1). in Mammalian CellsThese results suggest that p53and p63may have par-Having established the biological activities of p53in thetially overlapping roles in modulating the expression of frog embryo,we wished to determine to what extentat least one key gene in the TGF-␤cytostatic program p53is required for TGF-␤-mediated gene responses inin keratinocytes.human cells.We monitored the expression of a groupGiven the gene expression changes observed in the of genes playing key roles in different aspects of theresponse to Activin after ablation of p53in HepG2cells, cellular response to TGF-␤signaling in mammals,suchwe assayed for their biological importance in the context as p21WAF1,plasminogen activator inhibitor-1(PAI-1),of the TGF-␤cytostatic program.For this purpose,bro-matrix metalloprotease-2(MMP2),and TGF-␤induciblemodeoxyuridine(BrdU)incorporation was analyzed as early gene(TIEG).p21WAF1is a Cyclin-dependent kinasean indication of DNA synthesis and S phase entry of (CDK)inhibitor and a central mediator of the cellularcontrol and p53-depleted cells(Figure3D).As expected, growth arrest program(Deng et al.,1995).PAI-1andActivin inhibited cell-cycle progression of control HepG2 MMP2are secreted proteins required for extracellularcells.Strikingly however,Activin had limited effect on matrix remodeling and epithelial-mesenchymal transfor-the BrdU incorporation after siRNA-mediated ablation mation,whereas TIEG is thought to be important in TGF-of p53,indicating that p53knockdown is sufficient to ␤-mediated apoptosis.overcome the growth arrest imposed by TGF-␤sig-We analyzed the relevance of p53in the activation ofnaling.these TGF-␤targets.For this purpose,we reduced theLack of sensitivity to TGF-␤growth-suppressing ef-endogenous levels of p53using the small interferingfects is a landmark of most cancers.Given that a high RNA(siRNA)technique in the HepG2model system(Fig-proportion of human cancers carry mutations in p53, ure3A),as this hepatoma cell line is highly TGF-␤/Activinour data at least suggest that inactivation of p53may responsive and expresses wild-type p53,but not p63be one of the possible mechanisms for the selective or p73.Figure3B shows RT-PCR analyses in which allloss of TGF-␤tumor-suppressing effects in cancer cells. the markers were clearly upregulated in HepG2cells asWe explored this hypothesis trying to restore the anti-an early response to Activin treatment.In the presenceproliferative effects of TGF-␤in cancer cell lines lacking of anti-p53siRNA,these inductions were reduced forp53.SAOS-2is a p53null osteosarcoma cell line,not p21WAF1,MMP2,and PAI-1.Notably,induction of TIEGexpressing p63or p73,that is insensitive to growth ar-was p53independent,implicating that p53knockdownrest mediated by TGF-␤treatment or overexpressed has no effect,per se,on the overall TGF-␤respon-Smad2(Figure3E,lane2,and data not shown)(Prunier siveness.This further suggests that knockdown of p53et al.,2001).Strikingly,however,coexpression of Smad2 affects a significant proportion,but not all the TGF-␤and low amounts of p53AS,by themselves unable to target genes,as previously noted in the TGF-␤differenti-trigger any effect,resulted in a marked inhibition of cell ation program of the frog embryo.growth as measured by BrdU incorporation(compare The CDK inhibitor p21WAF1is a critical determinant oflane5and6).Thus,reintroduction of p53activity in p53-growth arrest in response to a variety of stimuli;thesedeficient cancer cells may restore part of the TGF-␤activate p21WAF1expression p53dependently,such ascontrol over the cell cycle.DNA damage,or p53independently,such as terminaldifferentiation and senescence(Macleod et al.,1995).As our data argued that Activin/TGF-␤signaling and p53p53Is Required for TGF-␤1-Mediated Growth elevate in concert p21WAF1expression in HepG2cells,Arrest in Mouse Embryonic Fibroblastswe examined in more detail the effect of p53depletion and Hematopoietic Progenitorson the induction of p21WAF1by Activin at the protein level.Since transient siRNA ablation of p53had a strong im-Mock and anti-p53siRNA transfected HepG2cells were pact on the TGF-␤response of HepG2cells,we next stimulated for different times with Activin and then ana-asked whether the genetic inactivation of p53was also lyzed by Western blotting.As shown in Figure3C,the relevant for some biological responses to TGF-␤in nor-elevation of p21WAF1as response to Activin requires p53mal cell types.One complicating issue in the interpreta-Cell306Figure3.p53Activity Is Required for Full TGF-␤Responsiveness in Human Cells(A)Effectiveness of siRNA-mediated depletion of p53in HepG2cells controlled by Western blotting.(B)Control and p53-depleted HepG2cells were treated with Activin(1nM)for2hr and subjected to RT-PCR analyses for endogenous targets of Activin/TGF-␤.(C)Time course of p21WAF1induction by Activin(1nM)in mock and p53-depleted HepG2cells.(D)p53is required for Activin-mediated growth inhibition in HepG2cells treated with increasing doses of Activin for24hr.Columns show the effects of Activin expressed as percentage of inhibition of BrdU incorporation relative to unstimulated cultures.(E)p53rescues Smad2growth suppressing activity in p53null SAOS-2cells.Cells were transfected with the indicated plasmids and then plated onto coverslips for BrdU incorporation assay.Transfection of p53AS expression vector at high doses(lane3,750ng)blocked cell growth,but less than10-fold lower doses of p53AS had no effect(lane4,150ng;lane5,30ng).Cotransfection of of p53AS and Smad2(lane 6,30ng and1␮g,respectively)inhibited BrdU incorporation(up to87%inhibition).Arrows indicate the comparable amounts of p53AS.tion of genetic analyses is that the three p53family as judged by the decreased number of cells in S phase, members are coexpressed in most tissues in vivo(Yangthe concomitant increase in the G1phase and blockade and McKeon,2000).Nonetheless,recent data by Flores of BrdU incorporation.In contrast,p53Ϫ/ϪMEFs were et al.(2002)unveiled that induction of p21WAF1by DNAlargerly insensitive to TGF-␤1antiproliferative proper-damage relies on p53and is independent from p63and ties.As a control,we verified that the TGF-␤signalingcascade itself was effective in p53-deficient cells.For p73in mouse embryonic fibroblasts(MEFs).Thus,p53may be the main member of its family regulating p53-this purpose,we monitored the TGF-␤1-dependent tran-dependent growth arrest in this cell type.This simpli-scription of two synthetic reporters for Smad activation, fying finding provided us with a window of opportunity ARE3-lux and CAGA12-lux(Dennler et al.,1998;Huang to test a genetic requirement of p53for TGF-␤mediatedet al.,1995)and found that their inducibility was indistin-growth suppression.Primary wild-type and p53Ϫ/ϪMEFs guishable in wild-type and mutant cells(Figure4E).We were seeded at low density and treated with differentconclude that p53is a significant player in the antiprolif-doses of TGF-␤1;the distribution of the cell population erative effects mediated by TGF-␤in embryonic fibro-in the G1and S phases of the cell cycle was analyzedblasts.by flow cytometry after TGF-␤1treatment and the per-We next aimed to extend these findings to an addi-centage of cells in active DNA synthesis was assayedtional experimental system reflecting a role of TGF-␤in by BrdU incorporation.As shown in Figures4A–4D,wild-vivo.TGF-␤signaling has been shown to restrain the type MEFs were efficiently growth arrested by TGF-␤1,proliferative potential of hematopoietic progenitors。

EnMT

2008 Nov;135(21):3611-22. Epub 2008 Oct 2.LinksZhang J, Lin Y, Zhang Y, Lan Y, Lin C, Moon AM, Schwartz RJ, Martin JF, Wang F.Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W. Holcombe Boulevard, Houston, TX 77030, USA.The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2alpha (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2alpha in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2alpha mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.PMID: 18832393 [PubMed - in process]2008 Jul 28;182(2):315-25.LinksNiessen K, Fu Y, Chang L, Hoodless PA, McFadden D, Karsan A. Department of Medical Biophysics, British Columbia Cancer Agency, Vancouver V5Z 1L3, Canada.Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressedby a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergisticup-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.PMID: 18663143 [PubMed - indexed for MEDLINE]Potenta S, Zeisberg E, Kalluri R.[1] 1Division of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA [2] 2Department of Cell Biology, Harvard Medical School, Boston, MA, USA.Recent evidence has demonstrated that endothelial-to-mesenchymal transition (EndMT) may have a significant role in a number of diseases. Although EndMT has been previously studied as a critical process in heart development, it is now clear that EndMT can also occur postnatally in various pathologic settings, including cancer and cardiac fibrosis. During EndMT, resident endothelial cells delaminate from an organised cell layer and acquire a mesenchymal phenotype characterised by loss of cell-cell junctions, loss of endothelial markers, gain of mesenchymal markers, and acquisition of invasive and migratory properties.Endothelial-to-mesenchymal transition -derived cells are believed to function as fibroblasts in damaged tissue, and may therefore have an important role in tissue remodelling and fibrosis. In tumours, EndMT is an important source of cancer-associated fibroblasts (CAFs), which are known to facilitate tumour progression in several ways. These new findings suggest that targeting EndMT may be a novel therapeutic strategy, which is broadly applicable not only to cancer but also to various other disease states.British Journal of Cancer advance online publication, 16 September 2008;doi:10.1038/sj.bjc.6604662 .PMID: 18797460 [PubMed - as supplied by publisher]2008 Jul;24(4):462-8.LinksRieder F, Fiocchi C.Department of Internal Medicine I, University of Regensburg, Regensburg, Germany.PURPOSE OF REVIEW: Intestinal fibrosis is a potentially serious complication of inflammatory bowel disease and its pathophysiology is still unclear. This review will discuss recent developments relating to sources of fibroblasts in intestinal inflammation, mediators that modulate fibroblast activation and function, as well as new clinical, laboratory, endoscopic and radiological studies aimed at improving diagnosis and management of intestinal fibrosis in inflammatory bowel disease. RECENT FINDINGS: The fibroblast remains the central cell responsible for intestinal fibrosis in inflammatory bowel disease and transforming growth factor-beta1 is still the most potent pro-fibrogenic cytokine. Novel mediators, however, are being identified that modulate fibroblast function, such as interleukin-13, interleukin-21, galectin-3, osteopontin, Wnt and toll-like receptor ligands, and anti-tumor necrosis factor-alpha agents. New fibroblast sources are being identified, such as fibrocytes, and new mechanisms of fibroblast generation, like epithelial- and endothelial-to-mesenchymal transition. Animal models of intestinal fibrosis are still few, but new ways to induce gut fibrosis are being explored. Serological markers indicating a clinically complicated course that includes intestinal fibrosis are promising and are being tested in adult and pediatric populations, particularly in Crohn's disease. Video capsule endoscopy, the Given Patency capsule, double balloon enteroscopy, and computed tomographic enteroscopy are some of the new modalities being developed to assess the risk and improve the diagnosis of intestinal fibrosis. Novel therapeutic approaches include endoscopic balloon dilatation with conventional and double balloon enteroscopy, and local injection of glucocorticoids and tumor necrosis factor-alpha blockers, showing partial but encouraging success. SUMMARY: More studies are needed to improve knowledge of the pathophysiology of intestinal fibrosis if better preventive, diagnostic and therapeutic measures are to be expected in the near future.PMID: 18622160 [PubMed - indexed for MEDLINE]Nat Med. 2007 Aug;13(8):952-61. Epub 2007 Jul 29.LinksZeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, Gustafsson E, Chandraker A, Yuan X, Pu WT, Roberts AB, Neilson EG, Sayegh MH, Izumo S, Kalluri R.Division of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center & Harvard Medical School, Boston, Massachusetts 02215, USA.Cardiac fibrosis, associated with a decreased extent of microvasculature and with disruption of normal myocardial structures, results from excessive deposition of extracellular matrix, which is mediated by the recruitment of fibroblasts. The source of these fibroblasts is unclear and specificanti-fibrotic therapies are not currently available. Here we show that cardiac fibrosis is associated with the emergence of fibroblasts originating from endothelial cells, suggesting an endothelial-mesenchymal transition (EndMT) similar to events that occur during formation of the atrioventricular cushion in the embryonic heart. Transforming growth factor-beta1 (TGF-beta1) induced endothelial cells to undergo EndMT, whereas bone morphogenic protein 7 (BMP-7) preserved the endothelial phenotype. The systemic administration of recombinant human BMP-7 (rhBMP-7) significantly inhibited EndMT and the progression of cardiac fibrosis in mouse models of pressure overload and chronic allograft rejection. Our findings show that EndMT contributes to the progression of cardiac fibrosis and that rhBMP-7 can be used to inhibit EndMT and to intervene in the progression of chronic heart disease associated with fibrosis.PMID: 17660828 [PubMed - indexed for MEDLINE]2006 Jul;74(6):277-92.LinksArciniegas E, Neves YC, Carrillo LM.Servicio Autónomo Instituto de Biomedicina, Facultad de Medicina,Universidad Central de Venezuela, Apartado de correos 4043, Carmelitas,Caracas 1010, Venezuela. earciniegasbeta@Endothelial-to-mesenchymal transition (EndoMT) is a process throughwhich certain subsets of endothelial cells lose endothelial characteristicsand transform into mesenchymal or smooth muscle-like cells. Emergingevidence suggests that this process plays an important role during vasculardevelopment and in many vascular pathologies. As inepithelial-mesenchymal transition, EndoMT seems to progress through aseries of important steps whose interdependence and order are not clear, andthat some of them are regulated by soluble growth factors. Insulin-likegrowth factor II (IGFII), apart from being considered important in cancer,angiogenesis, and atherosclerotic lesions, is also considered as essential toembryonic development. Here, we report that addition of IGFII promotedthe EndoMT process in the presence of very low amounts of chicken serumto arrested primary embryonic aortic chicken endothelial cells attached tofibronectin (FN), gelatin, or native type I collagen. This was demonstratedby cell spreading, loss of cell-cell contacts, detachment, migration, andtransformation. These cellular events also occurred when IGFII was addedto medium containing vitronectin (VN). Additionally, we demonstrated thatthese proteins were present in the spontaneous intimal thickenings that areobserved at day 11-13 of chicken embryo development. We also show thatalterations in the distribution of VE-cadherin and beta-catenin occur afterIGFII and serum or VN stimulation, and propose that the via VN IGFIIeffects may be facilitated by interaction of the mannose-6-phosphate/IGFIIreceptor (M6P/IGFIIR) with the urokinase-type plasminogen activatorreceptor (uPAR) and its ligand (uPA). Collectively, these findings providethe first evidence for a potential role of the IGFII-VN complex during theEndoMT process. From our observations and previous studies, we postulatea working hypothesis supporting a fundamental role for these moleculesduring EndoMT.PMID: 16831197 [PubMed - indexed for MEDLINE]Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8Hironobu Fujiwara a, b, Jianguo Gu a and Kiyotoshi Sekiguchi a, b, ,a Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japanb Sekiguchi Biomatrix Signaling Project, ERATO, Japanese Science and Technology Corporation, Aichi Medical University, Nagakute-cho, Aichi480-1195, JapanReceived 6 February 2003;revised 1 July 2003.Available online 3 October 2003.AbstractBlood vessel formation requires endothelial cell interactions with the extracellular matrix through cell surface receptors, and signaling events that control endothelial cell adhesion, migration, and lumen formation. Laminin-8 (α4β1γ1) is present in all basement membranes of blood vessels in fetal and adult tissues, but despite its importance in vessel formation, its role in endothelial cell adhesion and migration remains undefined. We examined adhesion and migration of HMEC-1 human microvascular endothelial cells on laminin-8 with an emphasis on the integrin-mediated signaling events, as compared with those on laminin-10/11 and fibronectin. We found that laminin-8 was less potent in HMEC-1 cell adhesion than laminin-1, laminin-10/11, and fibronectin, and mediated cell adhesion through α6β1 integrin. Despite its weak cell-adhesive activity, laminin-8 was as potent as laminin-10/11 in promoting cell migration. Cells adhering to laminin-8 displayed streaks of thin actin filaments and formed lamellipodia at the leading edge of the cells, as observed with cells adhering to laminin-10/11, while cells on fibronectinshowed thick actin stress fibers and large focal adhesions. Pull-down assays of GTP-loaded Rho, Rac, and Cdc42 demonstrated that Rac, but not Rho or Cdc42, was preferentially activated on laminin-8 and laminin-10/11, when compared with fibronectin. Furthermore, a dominant-negative mutant of Rac suppressed cell spreading, lamellipodial formation, and migration on laminin-8, but not on fibronectin. These results, taken together, indicate that Rac is activated during endothelial cell adhesion to laminin-8, and is pivotal for α6β1 integrin-mediated cell spreading and migration on laminin-8.Author Keywords: Basement membrane; Laminin; Endothelial cell; Integrin; RacAbbreviations: FBS, fetal bovine serum; HUVECs, human umbilical vein endothelial cells; mAb, monoclonal antibody; PBS, phosphate-buffered saline; GST-RBD, a fusion protein of glutathione S-transferase to the Rho-binding domain of rhotekin; GST-CRIB, a fusion protein of glutathione S-transferase to the Cdc42/Rac-interactive-binding domain of PAK1; BSA, bovine serum albuminArticle Outline• Introduction• Materials and methods• Cell culture• Reagents and antibodies• Cell-adhesive proteins• Purification of laminin-8• SDS-PAGE and immunoblotting• Expression vectors• Cell spreading assay• Cell migration assay and microinjection• Immunofluorescence staining• Detection of GTP-loaded Rho, Rac, and Cdc42• Results• HMEC-1 cell adhesion to laminin-8• Laminin-8 stimulates HMEC-1 migration through α6β1 inTransdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle-like cells regulated by myocardin involved in hypoxia-induced pulmonary vascular remodelling.P Zhu, L Huang, X Ge, F Yan, R Wu, and Q AoInt J Exp Pathol, December 1, 2006; 87(6): 463-74.AbstractF ull text via InfotrieveAlert me when citedF ind more like thisDepartment of Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan City, China.Myocardin gene has been identified as a master regulator of smooth muscle cell differentiation. Smooth muscle cells play a critical role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodelling (PVR). The purpose of this study was to investigate the change of myocardin gene expression in the pulmonary vessels of hypoxia-induced PH affected by Sildenafil treatment and the involvement of endothelial cells transdifferentiation into smooth muscle cells in the process of hypoxia-induced PH and PVR. Myocardin and relative markers were investigated in animal models and cultured endothelial cells. Mean pulmonary artery pressure (mPAP) was measured. Immunohistochemistry and immunofluorescence were used to show the expression of smooth muscle alpha-actin (SMA), in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT-PCR) were performed respectively to detect the myocardin and SMA expression at mRNA levels. Small interfering RNA (siRNA) induced suppression of myocardin in cultured cells. We confirmed that hypoxia induced the PH and PVR in rats. Sildenafil could attenuate thehypoxia-induced PH. We found that myocardin mRNA expression is upregulated significantly in the hypoxic pulmonary vessels and cultured cells but downregulated in PH with Sildenafil treatment. The porcine pulmonary artery endothelial cells (PAECs) transdifferentiate into smooth muscle-like cells in hypoxic culture while the transdifferentiation did not occur when SiRNA of myocardin was applied. Our results suggest that myocardin gene, as a marker of smooth muscle cell differentiation, was expressed in the pulmonary vessels in hypoxia-induced PH rats, which could be downregulated by Sildenafil treatment, as well as in hypoxic cultured endothelial cells. Hypoxia induced the transdifferentiation of endothelial cells of vessels into smooth muscle-like cells which was regulated by myocardin.Erratum in Int J Exp Pathol. 2007 Apr;88(2):127-8Publication Types。

中风醒脑液SD大鼠含药血清对体外培养神经细胞缺血再灌注损伤保护作用的实验研究_李艳青

中风醒脑液SD 大鼠含药血清对体外培养神经细胞缺血再灌注损伤保护作用的实验研究李艳青1，张晓云2（1.成都中医药大学，四川成都610075；2.成都中医药大学附属医院，四川成都610072）摘要：目的：探讨中药复方中风醒脑液SD 大鼠含药血清对体外培养的神经细胞缺血再灌注损伤的保护作用，为进一步开展临床研究提供依据。

方法：采用血清药理学和原代神经细胞培养的方法，用SD 大鼠的含中风醒脑液血清体外培养PC －12神经细胞，用连二亚硫酸钠造成细胞缺氧损伤模型，用H 2O 2造成细胞氧化损伤模型，建立缺血再灌注的体外细胞模型，分别在造模后2h 、4h 用alamarBlue 染色，用酶标仪测定吸光度，检测PC －12细胞的生长活力及应用流式细胞技术检测细胞的凋亡率。

结果：中药复方中风醒脑液SD 大鼠含药血清对体外培养的神经细胞缺血再灌注损伤具有保护作用。

结论：中风醒脑液是一种神经细胞保护剂，可以增加神经细胞存活率与活力，抑制其凋亡率。

关键词：中风醒脑液；缺血再灌注；实验研究中图分类号：R255．2文献标识码：B 文章编号：1000－1719（2011）10－2089－03收稿日期：2011－02－16基金项目：国家自然科学基金项目（30973747）作者简介：李艳青（1980－），女，山东潍坊人，博士研究生，研究方向：中西医结合内科学。

通讯作者：张晓云（1953－），女，四川乐山人，教授、主任医师，博士研究生导师，研究方向：中西医结合内科学。

缺血性脑血管病（Ischemic Cerebral Vesscular Dis-ease ，ICVD ）是威胁人类健康与生存的主要疾病之一，致残率极高，是目前重点防治的疾病之一。

在ICVD 的治疗中重建血流或增强缺血区的血流供应是缺血脑组织修复损伤的必需条件，但同时带来的再灌注损伤也是目前最受关注的问题。

全国重点专病“中风病”建设单位、成都中医药大学附属医院急诊科陈绍宏教授是我国著名中医急症专家，他经过多年潜心观察和临床验证，提出了“中风核心病机”理论［1］，认为中风病的核心病机是：元气亏虚、痰瘀互阻、风火相煽；其中以元气虚为本，痰、瘀、风、火都是继发于元气虚的内生之邪。

癌症生物学(英文版)

11
Resisting cell death Sensors that trigger apoptosis:
• DNA damage sensor functions via p53 • Insufficient survival factor signaling (e.g. IL-3 for lymphocytes) • Hyperactive signaling by some oncoproteins (e.g. Myc) • Each of these can activate BH3-only proteins to induce apoptotic
• Loss of RB function (by deletion or mutation) removes a gatekeeper of cell cycle progression resulting in persistent cell proliferation
• p53 protein senses intracellular stress and abnormality • If DNA damage is present or if growth promoting signals, oxygen or
and inactivating two pro-apoptotic proteins, Bax and Bak, that live in the mitochondrial membrane • Bax and Bak, when released from Bcl-2 binding (and inhibition), disrupt the outer mitochondrial membrane, releasing cytochrome c which activates the cascade of proteolytic capsases leading to the cellular changes of apoptosis • Bcl-2 interacts with Bax and Bak via BH3 interaction domains • Other proteins that sense cellular abnormalities contain BH3 domains (“BH3-only” proteins) can activate apoptosis by interfering with Bcl-2 or by activating Bax or Bak directly

成骨分化相关信号通路的研究进展

Advances in Clinical Medicine 临床医学进展, 2017, 7(4), 235-241Published Online October 2017 in Hans. /journal/acmhttps:///10.12677/acm.2017.74039Research Progress of Osteogenesis-Related Signaling PathwaysLei Zhou, Minghai Wang*Department of Orthopedics, The Fifth People’s Hospital of Shanghai, Fudan University,ShanghaiReceived: Sep. 27th, 2017; accepted: Oct. 7th, 2017; published: Oct. 16th, 2017AbstractObjective: Osteogenesis is the foundation of bone formation and key procedure of bone metabol-ism. In recent years, major progress was made in the molecular mechanism of osteogenesis at home and abroad. Therefore, the mechanism and research progress of osteogenesis-related sig-naling pathways was reviewed. Methods: Literature about ossification and osteogenesis-relate signaling pathways in recent years were reviewed and analyzed. Results: Several signaling path-ways have been found osteogenesis-related, among them, BMP-SMAD, Wnt/β-Catenin, Notch, Hedgehog, MAPK and FGF signaling pathways play the leading role in bone-formation. Besides, a complex regulatory network is composed of interactions between multiple signaling pathways.However, the specific mechanism of osteogenesis-related signaling pathways is still unclear be-cause of limited research methods. Conclusion: To make clear the mechanism of these signaling pathways respectively and their interactions is of great significance for illustrating the complete mechanism of osteogenesis.KeywordsBone Metabolism, Osteogenesis, Signaling Pathway成骨分化相关信号通路的研究进展周雷，王明海*复旦大学附属上海市第五人民医院骨科，上海收稿日期：2017年9月27日；录用日期：2017年10月7日；发布日期：2017年10月16日*通讯作者。

Invitrogen Neon

Neon™ Transfection SystemFor transfecting mammalian cells, including primary and stem cells, with high transfection efficiency. Catalog Numbers MPK5000, MPK1025, MPK1096, MPK10025, MPK10096Doc. Part No. 25-1056 Pub. No. MAN0001632 Rev.B.0WARNING! For safety and biohazard guidelines, see the “Safety” appendix in the Neon™ Transfection System User Guide (Pub.No. MAN0001557). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.Note: This Quick Reference is intended as a benchtop reference for experienced users of the Neon™ Transfection System User Guide (Pub. No. MAN0001557). For detailed instructions, supplemental procedures, and troubleshooting, see the Neon™ Transfection System User Guide (Pub. No. MAN0001557).General guidelines•Prepare high-quality plasmid DNA at a concentration of 1 to 5 μg/μL in deionized water or TE buffer, or high quality RNAi duplex at a concentration of 100–250 μM in nuclease-free water.•Use an appropriate GFP (green fluorescent protein) construct or siRNA control to determine transfection efficiency. See the Neon™Transfection System User Guide (Pub. No. MAN0001557) for details.•Use Resuspension Buffer R for established adherent and suspension cells, as well as primary adherent cells. Use Resuspension Buffer T with high voltage protocols of 1900 V or more. If arcing occurs with Resuspension Buffer R, consider switching toResuspension Buffer T.•Based on your initial results, you may need to optimize the electroporation parameters for your experiment using an 18-well or pre-programmed 24-well optimization protocol.•Discard the Neon™ Tips after 2 usages and Neon™ Tubes after 10 usages as a biological hazard. Change the tube and buffer when switching to a different plasmid DNA/siRNA or cell type.•The volume of plasmid DNA or siRNA added to the tranfection reaction should not exceed 10% of the total transfection volume.•Visit for a library of electroporation protocols for a variety of commonly used cell types.Prepare cellsFor the appropriate volume of medium to use based on cell density, or plating volumes for other plate formats, see “Amount of reagents” on page 2.1.Cultivate the required number of cells (70% to 90%confluent on the day of transfection) by seeding a flaskcontaining fresh growth medium 1 to 2 days prior toeletroporation.2.On the day of the experiment, pre-warm aliquots of culturemedium containing serum, PBS (without Ca2+and Mg2+), and Trypsin/EDTA solution to 37°C.3.Rinse the cells with PBS (without Ca2+and Mg2+), thentrypsinize the cells with the Trypsin/EDTA solution.4.Take an aliquot of trypsinized cell suspension, then countcells to determine the cell density.5.Harvest the cells in growth medium containing serum.6.Transfer cells to a 1.5-mL microcentrifuge tube or a 15-mLconical tube, then centrifuge the cells at 100 - 400 × g for 5 minutes at room temperature.7.Wash cells with PBS (without Ca2+and Mg2+) bycentrifugation at 100 - 400 × g for 5 minutes at roomtemperature.8.Aspirate the PBS, then resupsend the cell pellet inResuspension Buffer R (or Resuspension Buffer T forprograms ≥ 1900 V) at a final density of 1.0 × 107 cells/mL for adherent cells or 2.0 × 107 cells/ml for suspension cells.Gently pipette the cells to obtain a single cell suspension.IMPORTANT! Avoid storing the cell suspension for more than 15 to 30 minutes at room temperature. This will reduce cell viability and transfection efficiency.9.Prepare 24-well plates by filling the wells with 500 μL ofculture medium containing serum and supplements, butwithout antibiotics. Pre-incubate plates in a 37°C, 5% CO2 humidified incubator.Amount of reagentsFor each electroporation sample, the amount of plasmid DNA/siRNA, cell number, and volume of plating medium per well are listed in the following table. Use Resuspension Buffer T for cell types that require high voltage protocols of 1900 V or more. For all other cell types, use Resuspension Buffer R.[1]Use Resuspension Buffer T for primary suspension blood cells.Using the Neon ™Transfection SystemFor details on setting up the Neon ™device and Neon ™PipetteStation, see the Neon ™Transfection System User Guide (Pub. No.MAN0001557).1.Select the appropriate protocol for your cell type. Use one ofthe following options:•Input the electroporation parameters in the Input window if you already have the electroporation parameters for your cell type.•Tap Database , then select the cell-specificelectroporation parameters that you have added for various cell types.•Tap Optimization to perform the optimization protocol for your cell type.2.Fill the Neon ™Tube with 3 mL of Electrolytic Buffer (useBuffer E for the 10 μL Neon ™Tip and Buffer E2 for the 100μL Neon ™Tip).Note: Make sure that the electrode on the side of the tube is completely immersed in buffer. 3.Insert the Neon ™ Tube into the Neon ™Pipette Station untilyou hear a click sound (Figure 1).Figure 1 Schematic of Neon ™ Tube and Neon ™ Pipette Station.4.Transfer the appropriate amount of plasmid DNA/siRNA intoa sterile, 1.5 mL microcentrifuge tube.5.Add cells to the tube containing plasmid DNA/siRNA, thengently mix. See “Amount of reagents” on page 2 for cell number, DNA/siRNA amount, and plating volumes to use.6.To insert a Neon ™Tip into the Neon ™Pipette, press thepush-button on the pipette to the second stop to open the clamp.7.Insert the top-head of the Neon ™Pipette into the Neon ™Tipuntil the clamp fully picks up the mount stem of the piston (Figure 2).Figure 2 Schematic of Neon ™ Pipette and Neon ™Tip.8.Gently release the push-button, continuing to apply adownward pressure on the pipette, ensuring that the tip is sealed onto the pipette without any gaps.9.Press the push-button on the Neon ™Pipette to the first stopand immerse the Neon ™Tip into the cell-DNA/siRNA mixture.Slowly release the push-button on the pipette to aspirate thecell-DNA/siRNA mixture into the Neon ™Tip (Figure 3).Figure 3 Schematic of Neon ™Tip.Note: Avoid air bubbles during pipetting as air bubbles cause arcing during electroporation leading to lowered or failed transfection. If you notice air bubbles in the tip,discard the sample, then carefully aspirate the fresh sample into the tip again without any air bubbles.10.Insert the Neon ™Pipette with the sample vertically into theNeon ™ Tube placed in the Neon ™Pipette Station until youhear a click sound (Figure 4).Figure 4 Schematic of Neon ™ Tube and Neon ™ Pipette Station.Note: Ensure that the metal head of the Neon ™pipette projection is inserted into the groove of the pipette station.11.Ensure that you have selected the appropriateelectroporation protocol, then press Start on the touchscreen.12.The Neon ™device automatically checks for the properinsertion of the Neon ™ Tube and Neon ™Pipette before delivering the electric pulse.13.After delivering the electric pulse, Complete is displayed onthe touchscreen to indicate that electroporation is complete.14.Slowly remove the Neon ™Pipette from the Neon ™PipetteStation. Immediately transfer the samples from the Neon ™Tip by pressing the push-button on the pipette to the first stop into the prepared culture plate containing prewarmed medium with serum and supplements but without antibiotics.Note: Discard the Neon ™ Tip into an appropriate biologicalhazardous waste container. To discard the Neon ™Tip, press the push-button to the second stop into an appropriate biological hazardous waste container.15.Repeat step 6 to step 14 for the remaining samples.Note: Be sure to change the Neon ™Tips after using it twiceand Neon ™ Tubes after 10 usages. Use a new Neon ™Tip andNeon ™Tube for each new plasmid DNA sample.16.Gently rock the plate to ensure even distribution of the cells.Incubate the plate at 37℃ in a humidified CO 2 incubator.17.If you are not using the Neon ™device, turn the power switchon the rear to OFF .18.Assay samples to determine the transfection efficiency(e.g., fluorescence microscopy or functional assay) or geneknockdown (for siRNA).19.Based on your initial results, you may need to optimizedthe electroporation parameters for your cell type. For more information, see the Neon™ Transfection System User Guide (Pub. No. MAN0001557).Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USAFor descriptions of symbols on product labels or product documents, go to /symbols-definition.The information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified./support | /askaquestion。

成纤维细胞生长因子的研究进展

成纤维细胞生长因子的研究进展纪孝伟;吴春玲【摘要】成纤维细胞生长因子(FGF)在机体内广泛分布,其系统由多个受体和配体组成,在组织、神经修复和血管再生中起到了重要作用,对多种细胞有促进DNA和细胞分裂的作用.FGF系统在脑内的作用主要是调控神经元、神经胶质细胞和血管内皮细胞的增殖、移行、分化和存活.随着分子生物学等技术的不断进步,人们对FGF的研究不断深入,FGF有望成为一种新的治疗手段,为疾病的治疗提供新思路.【期刊名称】《医学综述》【年(卷),期】2014(020)003【总页数】3页(P411-413)【关键词】成纤维细胞生长因子;神经修复;内脏发育【作者】纪孝伟;吴春玲【作者单位】山东中医药大学第二附属医院神经内科,济南,250001;山东大学附属济南市中心医院肾脏病/血液净化中心,济南,250013【正文语种】中文【中图分类】R741.02成纤维细胞生长因子(fibroblast growth factor，FGF)是一种能调节细胞增殖、分化的多肽家族，其系统由多个受体和配体组成，在机体内的许多组织和器官内均有分布。

其中以碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和酸性成纤维生长因子的研究最为活跃。

FGF是一种多功能的非特异性活跃物质，对来源于中胚层和神经外胚层的多种细胞有促进DNA和细胞分裂的作用，并且可以延缓细胞的衰老。

该文就FGF的组成、分布、功能及其临床作用予以综述。

1 FGF系统的组成和分布人类FGF系统有22个配体和5个受体组成。

成纤维细胞生长因子受体(fibroblast growth factor receptor，FGFR)属于酪氨酸激酶受体家族，在细胞外的第三个免疫球蛋白样区可以被剪切成不同的突变体。

在海马的CA2和CA3区FGFR1表达量最高，在皮质表达则相对较少。

与FGFR1的分布不同，FGFR2和FGFR3在海马星形细胞的表达量少[1]。

每日专业名词通俗解释

每⽇专业名词通俗解释2022年03⽉01⽇HSCR⽂章的⼀些科普progenitor和precursor的区别The main difference between progenitor and precursor cells is that progenitor cells are mainly multipotent cells that can differentiate into many types of cells, whereas precursor cells are unipotent cells that can only differentiate into a particular type of cells.糖酵解glycolysis糖酵解（英语：glycolysis，⼜称糖解）是把葡萄糖（C6H12O6）转化成丙酮酸（CH3COCOO− + H+）的代谢途径。

在这个过程中所释放的⾃由能被⽤于形成⾼能量化合物ATP和NADH。

糖酵解作⽤及其各种变化形式发⽣在⼏乎所有的⽣物中，⽆论是有氧和厌氧。

糖酵解的⼴泛发⽣显⽰它是最古⽼的已知的代谢途径之⼀。

糖酵解作⽤是所有⽣物细胞糖代谢过程的第⼀步。

糖酵解作⽤是⼀共有10个步骤酶促反应的确定序列。

在该过程中，⼀分⼦葡萄糖会经过⼗步酶促反应转变成两分⼦丙酮酸。

糖酵解作⽤发⽣在⼤多数⽣物体中的细胞的胞质溶胶。

脂肪酸分解Fatty acid catabolism脂肪酸的氧化作⽤發⽣在粒線體（mitochondria）內，脂肪酸必須先和ATP反應，轉變為活化的中間產物，才能與其他酵素作更進⼀步的代謝，⾧鏈的Fatty acyl-CoA不能穿過粒線體內膜進⼊粒線體基質，需藉⾁酸素（carnitine）運送機制，脂肪酸氧化(Fatty acid oxidation)。

氧化磷酸化（英语：oxidative phosphorylation，缩写作 OXPHOS）是细胞的⼀种代谢途径，该过程在真核⽣物的线粒体内膜或原核⽣物的细胞膜上发⽣，使⽤其中的酶及氧化各类营养素所释放的能量来合成三磷酸腺苷（ATP）。

Differentiation of Human Pluripotent Stem Cells into Retinal Cells

87M.A. Hayat (ed.), Stem Cells and Cancer Stem Cells, Volume 6,DOI 10.1007/978-94-007-2993-3_9, © Springer Science+Business Media B.V . 20129A bstractRetinal and macular degeneration disorders are characterized by a progressive loss of photoreceptors, which causes visual impairment and blindness. In some cases, the visual loss is caused by dysfunction, degen-eration and loss of underlying retinal pigment epithelial (RPE) cells and the subsequent death of photoreceptors. The grim reality is that there is no successful treatment for most of these blindness disorders. Cell therapy aimed at replenishing the degenerating cells is considered a potential ther-apeutic approach that may delay, halt or perhaps even reverse degenera-tion, as well as improve retinal function and prevent blindness in the aforementioned conditions. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs) may serve as an unlimited donor source of photoreceptors and RPE cells for transplantation into degenerat-ing retinas and for retinal disease modeling.I ntroductionThe vertebrate eyes form as bilateral evaginations of the forebrain, called optic vesicles (Martínez-Morales et al. 2004 ; Fig. 9.1a ). During develop-ment, the optic vesicles begin to invaginate to form a cup-shaped structure, the optic cup. The inner, thicker neural layer of the optic cup differ-entiates into the neural retina, and the outer, thin-ner pigmented layer forms the retinal pigmentepithelium (RPE). At the early developmental stages, the neuroepithelial cells that compose the optic vesicle are morphologically and molecu-larly identical and are all able to give rise to neu-ral retina and RPE. Exogenous signals coming from the adjacent tissues, including factors from the ﬁ broblast growth factor (FGF) and transform-ing growth factor beta (TGF b ) families, dictate the fate of these cells. The mature vertebrate ret-ina is comprised of six types of neurons and one type of glia (the Müller glia). These seven cell types constitute three nuclear layers: retinal gan-glion cells in the ganglion cell layer (GCL); the horizontal, bipolar and amacrine interneurons, and Müller glial cells in the inner nuclear layer (INL); and rod and cone photoreceptors in the outer nuclear layer (ONL; Harada et al. 2007;M . I delson • B . R eubinoff (*)T he Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy & The Department of Obstetrics and Gynecology , H adassah University Medical Center ,E in Kerem 12000 ,J erusalem 91120 ,I srael e -mail: b enjaminr@ekmd.huji.ac.il D ifferentiation of HumanPluripotent Stem Cells into Retinal Cells Masha Idelson and Benjamin Reubinoff88M. Idelson and B. ReubinoffFig. 9.1b ). The photoreceptor cells capture lightphotons and transform their energy into electrical signals by a mechanism called phototransduction. The visual pigment which is utilized in this process is located on membranal discs in the outer seg-ments of photoreceptors. The outer segments are continuously renewed: the old discs are shed and new disks form. When the photoreceptors absorb light, they send the signal through the retinal interneurons to the ganglion cells which transmit the electrical impulse to the brain by their axons forming the optic nerve. Rods are responsible for night vision, whereas cones are responsible for color vision and detecting ﬁ ne details. The macula is a small part of the retina which is rich in cones and responsible for detailed central vision.R PE cells that compose the outer layer of the optic cup are pigmented cuboidal cells which lie between the neural retina and the choriocapil-laris, which include the blood vessels supplying the retina. The multiple villi on their apical side are in direct contact with the outer segments ofextraocular mesenchymeabneural retinalensoptic nerveoptic cupsurface ectodermRPEFGFoptic vesiclechoroidBM RPE cone ONLINL GCLlightHC BC MC ACONrod F ig. 9.1 D evelopment and structural arrangement of the retina. ( a ) Schematic representation of retinal development including the transition from optic vesicle to optic cup and retinal patterning. ( b ) Schematic diagram of retinal cells arrangement and connections. A bbreviations :A C amacrinecell, B C bipolar cell, B M Bruch’s membrane, G CL gan-glion cell layer, H C horizontal cell, I NL inner nuclear layer, M C Müller cell, O N optic nerve, O NL outer nuclear layer89 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsthe photoreceptor cells; on their basal side, the RPE is in contact with the underlying basal mem-brane, termed Bruch’s membrane that separates the RPE from the choroid. These cells play cru-cial roles in the maintenance and function of the retina and its photoreceptors. As a layer of pig-mented cells, the RPE absorbs the stray light that was not absorbed by the photoreceptors. The RPE cells form a blood–retinal barrier due to decreased permeability of their junctions. The RPE cells transport ions, water, and metabolic end products from the retina to the bloodstream. They are involved in supplying the neural retina with nutrients from the bloodstream, such as glu-cose, retinol, and fatty acids. Another important function of the RPE is the phagocytosis of shed photoreceptor outer segments. After the outer segments are digested, essential substances such as retinal are recycled. Retinal is also recycled and returned to photoreceptors by the process known as the visual cycle. The precise functioning of the RPE is essential for visual performance. Failure of one of these functions can lead to degeneration of the retinal photoreceptors, vision impairment and blindness.T here are many inherited and age-related eye disorders that cause degeneration of the retina as a consequence of loss of photoreceptor cells. Retinal and macular degeneration disorders can be divided into two main groups. The ﬁ rst group primarily affects the photoreceptors and involves the majority of cases of retinitis pigmentosa. In the second group, the primary damage is to the adjacent RPE cells, and as a consequence of this damage, the photoreceptors degenerate. This group includes age-related macular degeneration, Stargardt’s macular dystrophy, a subtype of Leber’s congenital amaurosis in which RPE65 is mutated, Best’s disease and some cases of retini-tis pigmentosa, as well.W ith regard to retinitis pigmentosa (RP), it is a group of inherited retinal degeneration diseases that are caused, as mentioned above, by a primary progressive loss of rod and cone photoreceptors, followed by a subsequent degeneration of RPE (Hartong et al. 2006). The disease affects approxi-mately 1.5 million patients worldwide and is the most common cause of blindness in people under 70 years of age in the western world. The disease can be characterized by retinal pigment deposits visible on the fundus examination. In most cases, the disease primarily affects rods. At later stages of the disease, the degeneration of cones takes place. As a consequence of disease progression, the patients’ night vision is reduced. Patients initially lose peripheral vision while retaining central vision (a visual status termed “tunnel vision”). In advanced cases, central vision is also lost, commonly at about 60 years of age. The disease affects about 1 in 4,000. The inheritance can be autosomal-recessive, autosomal-dominant or X-linked (in ~50–60%, 30–40%, and 5–15% of cases, respectively). Mutations in more than 140 genes have been iden-tiﬁ ed as causing RP (Hartong et al. 2006).Among these genes are those involved in phototransduc-tion, like rhodopsin, the a- and b- subunits of phos-phodiesterase, the a- and b- subunits of Rod cGMP gated channel and arrestin. The additional muta-tions were found in genes encoding structural pro-teins, like peripherin, rod outer segment protein and fascin. They were also found in transcription factors involved in photoreceptors’ development such as Crx and Nrl, and in other genes, whose products are involved in signaling, cell-cell interac-tion and trafﬁcking of intracellular proteins. Currently, there is no effective cure for RP. Treatment with vitamin A palmitate, omega-3 fatty acids and other nutrients may somewhat slow the rate of the disease progression in many cases. Reduction in exposure to light was also shown to decrease the rate of retinal degeneration.A mong the group of retinal degenerations that are caused by primary loss of RPE cells or their function, age-related macular degeneration (AMD) is the most frequent condition and the leading cause of visual disability in the western world (Cook et al. 2008).Among people over 75 years of age, 25–30% are affected by AMD, with progressive central visual loss that leads to blindness in 6–8%. The retinal degeneration pri-marily involves the macula. The dry form of AMD is initiated by hyperplasia of the RPE and formation of drusen deposits, consisting of meta-bolic end products underneath the RPE or within the Bruch’s membrane. It may gradually progress into the advanced stage of geographic atrophy90M. Idelson and B. Reubinoff with degeneration of RPE and photoreceptorsover large areas of the macula causing central visual loss. Ten percent of dry AMD patients will progress to neovascular (wet) AMD, with blood vessels sprouting through the Bruch’s membrane with subsequent intraocular leakage and/or bleed-ing, accelerating the loss of central vision. While the complicating neovascularization can be treated with anti-VEGF agents, currently there is no effective treatment to halt RPE and photore-ceptor degeneration and the grim reality is that many patients eventually lose their sight (Cook et al. 2008).S targardt’s macular dystrophy (SMD) is the most common form of inherited macular dystro-phy affecting children (Walia and Fishman 2009). The disease is symptomatically similar to AMD. The prevalence of SMD is about 1 in 10,000 chil-dren. The disease involves progressive central visual loss and atrophy of the RPE beneath the macula following accumulation of lipofuscin in RPE cells, which is suggested to consist of non-degradable material, derived from ingested pho-toreceptor outer segments. The inheritance is predominantly autosomal recessive, although an autosomal dominant form has also been described. The mutation in the ABCA4 gene was found to be a most common cause of SMD. The product of the ABCA4 gene is involved in energy transport to and from photoreceptors. The mutated protein cannot perform its transport function and, as a result, photoreceptor cells degenerate and vision is impaired. Currently, there is no effective treat-ment for SMD.C ell therapy to replenish the degenerating cells appears as a promising therapeutic modality that may potentially halt disease progression in the various retinal and macular degeneration dis-orders caused by loss and dysfunction of RPE cells and photoreceptors (da Cruz et al. 2007).I n this chapter we will discuss the potential of human pluripotent cells which includes human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs), to gen-erate various types of retinal cells that could be used for transplantation therapy of retinal degen-eration disorders and disease modeling for drug discovery. C ell Therapy of Retinal and Macular DegenerationsT he eye is an attractive organ for cell therapy as it is easily accessible for transplantation and for simple monitoring of graft survival and potential complications by direct fundoscopic visualiza-tion. Anatomically, it is a relatively conﬁ ned organ limiting the potential of unwanted extra-ocular ectopic cell distribution, and a low number of cells are required to replenish the damaged cells. The eye is also one of the immune privi-leged sites of the body.T he concept of replacing dysfunctional or degenerated retina by transplantation has been developing ever since the ﬁ rst retina-to-retina transplant in 1986 (Turner and Blair 1986).In most studies, primary retinal immature (fetal) tissue has been used as donor material. It was demonstrated that such transplants can survive, differentiate, and even establish connections with the host retina to a limited degree (Ghosh et al. 1999). The subretinal transplantation of healthy RPE has some advantages over neural retinal transplantation, as it concerns only one cell type that is not involved in neural networking. Transplantation of RPE has been studied exten-sively in animal models (Lund et al. 2001).The most commonly used animal model of retinal degeneration is the Royal College of Surgeons (RCS) rat model, in which primary dysfunction of the RPE occurs as a result of a mutation in the receptor tyrosine kinase gene M ertk(D’Cruz et al. 2000). This leads to impaired phagocytosis of shed photoreceptor outer segments, with sec-ondary degeneration and progressive loss of pho-toreceptors within the ﬁ rst months of life. It was reported that rat and human RPE cells rescued photoreceptor cells from degeneration when transplanted into the subretinal space of RCS rats (Li and Turner 1988; Coffey et al. 2002).The ability of transplanted RPE cells to restore retinal structure and function has been demonstrated in clinical trials. In humans, autologous transplanta-tions of peripheral RPE as well as macular trans-locations onto more peripheral RPE provide a proof that positioning the macula above relatively91 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellshealthier RPE cells can improve visual functionin AMD patients (Binder et al. 2004; da Cruz et al. 2007). Nevertheless, the surgical procedures for autologous grafting are challenging and are often accompanied by signiﬁ cant complications. In addition, autologous RPE transplants may carry the same genetic background, environmen-tal toxic and aging-related effects that may have led to macular RPE failure and the development of AMD in the patient. It is also problematic to use autologous cells when all the RPE cells are damaged. Cell sources that can be used for such therapy include allogeneic fetal and adult RPE (Weisz et al. 1999; Binder et al. 2004; da Cruz et al. 2007). However, the use of fetal or adult retinal tissues for transplantation is severely lim-ited by ethical considerations and practical prob-lems in obtaining sufﬁcient tissue supply. The search for a cell source to replace autologous RPE such as immortalized cell lines, umbilical cord-derived cells as well as bone marrow-derived stem cells continues.T he derivation of hESCs more than a decade ago has raised immense interest in the potential clinical use of the cells for regeneration (Thomson et al. 1998; Reubinoff et al. 2000).Along the years, signiﬁ cant progress has been made towards the use of hESCs in clinical trials.T he other promising source of cells for transplantation therapy is iPSCs that are simi-lar to hESCs in their stemness characteristics and pluripotency. These cells could be gener-ated from different human somatic cells by transduction of four deﬁ ned transcription fac-tors: Oct3/4, Sox2, Klf4, and c-Myc (Takahashi et al. 2007).G eneration of RPE and neural retina from hESCs and iPSC has numerous advantages, as it can be done from pathogen-free cell lines under good manufacturing practice (GMP) conditions with minimal variation among batches. Such cells can be characterized extensively prior to preclinical studies or for clinical applications, and an unlimited numbers of donor cells can be generated from them. In the following para-graphs, strategies for induction of differentiation of hESCs and iPSCs towards RPE and neural retina fate are reviewed. D ifferentiation into Retinal Pigment EpitheliumI t was reported for the ﬁ rst time in mice and pri-mates that the differentiation of ES cells into RPE could be induced by co-culture with PA6 stromal cells (Kawasaki et al. 2002; Haruta et al. 2004). The resulting cells had polygonal epithelial mor-phology and extensive pigmentation. The cells expressed the markers that are characteristic of RPE. They developed typical ultrastructures and exhibited some functions of RPE. The differenti-ation of hESC into RPE was ﬁrst reported by Klimanskaya et al. (2004).According to their protocol, hESCs underwent spontaneous differ-entiation by overgrowth on mouse embryonic ﬁbroblasts (MEF), in feeder-free conditions or, alternatively, as embryoid bodies (EBs) in com-bination with withdrawal of bFGF from the medium. The yield of the formation of RPE cells after 4–8 weeks of spontaneous differentiation was relatively low; for example,<1% of EBs con-tained pigmented cells at this stage. However, after 6–9 months in culture, all the EBs contained pigmented cells. The areas of pigmented cells could be further isolated mechanically and prop-agated by passaging as RPE lines. Klimanskaya and colleges characterized the hESC-derived RPE cells by transcriptomics and demonstrated their higher similarity to primary RPE tissue than to human RPE lines D407 and ARPE-19. The low yield of spontaneously differentiating RPE cells was improved by induction of differentia-tion with Wnt and Nodal antagonists, Dkk1 and LeftyA, respectively, the factors that are sug-gested to promote retinal differentiation. This treatment gave rise to pigmented cells within 38% of the hESC colonies after 8 weeks (Osakada et al. 2008). Immunostaining with the ZO-1 anti-body showed that by day 120, hESC-derived pig-mented cells formed tight junctions (about 35% of total cells). We showed that differentiation toward the neural and further toward the RPE fate could be augmented by vitamin B3 (nicotin-amide; Idelson et al. 2009).We further showed that Activin A, in the presence of nicotinamide, efﬁ ciently induces and augments differentiation92M. Idelson and B. Reubinoffinto RPE cells. This is in line with the presumed role of Activin A in RPE development i n vivo .In the embryo, extraocular mesenchyme-secreted members of the TGF b superfamily are thought to direct the differentiation of the optic vesicle into RPE (Fuhrmann et al. 2000).Under our culture conditions, when the cells were grown in suspen-sion as free-ﬂ oating clusters, within 4 weeks of differentiation, 51% of the clusters contained pigmented areas and about 10% of the cells within the clusters were pigmented. When we modiﬁ ed the differentiation conditions to includea stage of monolayer culture growth, the yield of the RPE-like pigmented cells was signiﬁ cantly improved and 33% of the cells were pigmented after 6 weeks of differentiation. The derivation of RPE from hESCs and iPSCs without any external factor supplementation was also demonstrated by other groups (Vugler et al. 2008 ; Meyer et al. 2009 ; Buchholz et al. 2009).T he hESC-derived RPE cells were extensively characterized, including demonstration, both at the mRNA and the protein levels, of the expres-sion of RPE-speciﬁ c markers, such as RPE65, CRALBP, Bestrophin, Tyrosinase, PEDF, PMEL17, LRAT, isoforms of MiTF abundant in RPE, and others. The cells expressed markers of tight junctions that join the adjacent RPE cells: ZO-1, occludin and claudin-1 (Vugler et al. 2008 ) . Electron microscopic analysis revealed that the hESC-derived RPE cells showed features characteristic of RPE. The cells were highly polarized with the nuclei located more basally, and the cytoplasm with the mitochondria and melanin granules of different maturity more api-cally. A formation of basal membrane was observed on the basal surface of the RPE cell. Similar to putative RPE, the hESC-derived RPE basal membrane was shown to be composed of extracellular matrix proteins, collagen IV , lami-nin and ﬁ bronectin (Vugler et al.2008).The appearance of apical microvilli was demonstrated at the apical surface of the RPE. The presence of tight and gap junctions on the apical borders of the RPE cells was also conﬁ rmed by electron microscopy. O ne of the most important functions of RPE cells i n vivo is phagocytosis of shed photoreceptor outer segments, as part of the continuous renewal process of rods and cones. The hESC-derived RPE cells demonstrated the ability to phagocyto-size latex beads or puriﬁ ed photoreceptor outer segments, conﬁ rming that these cells are func-tionali n vitro . It may be concluded from all these studies that human pluripotent stem cells have a potential to give rise to pigmented cells exhibiting the morphology, marker expression and functionof authentic RPE.D ifferentiation into Retinal Progenitors and Photoreceptors O ur group showed, for the ﬁ rst time, the potential of highly enriched cultures of hESC-derived neu-ral precursors (NPs) to differentiate towards the neural retina fate (Banin et al. 2006).We demon-strated that the NPs expressed transcripts of key regulatory genes of anterior brain and retinal development. After spontaneous differentiation i n vitro , the NPs gave rise to progeny expressing markers of retinal progenitors and photoreceptor development, though this was uncommon and cells expressing markers of mature photorecep-tors were not observed. We showed that after transplantation into rat eyes, differentiation into cells expressing speciﬁ c markers of mature photoreceptors occurred only after subretinal transplantation (between the host RPE and pho-toreceptor layer) suggesting that this speciﬁ c microenvironment provided signals, yet unde-ﬁ ned, that were required to support differentia-tion into the photoreceptoral lineage.P rogress towards controlling and inducing the differentiation of hESCs into retinal progenitors and neurons i n vitro was reported in the study of Lamba et al. ( 2006).They treated hESC-derived EBs for 3 days with a combination of factors,including Noggin, an inhibitor of BMP signaling, Dkk1, a secreted antagonist of the Wnt signaling pathway and insulin-like growth factor 1 (IGF-1), which is known to promote retinal progenitor dif-ferentiation. The cultivation of EBs with these factors was followed by differentiation on Matrigel or laminin for an additional 3 weeks in the presence of the combination of the three93 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsfactors together with bFGF. Under these culture conditions, the majority of the cells developed the characteristics of retinal progenitors and expressed the speciﬁ c markers Pax6 and Chx10 (82% and 86% of the cells, respectively). The authors showed that after further differentiation, the cells expressed markers of photoreceptor development Crx and Nrl (12% and 5.75%, respectively). About 12% of the cells expressed also HuC/D, the marker of amacrine and ganglion cells. The expression of markers of the other sub-types of retinal neurons was demonstrated, as well. However, only very few cells (<0.01%) expressed markers of mature photoreceptors, blue opsin and rhodopsin. The abundance of cells expressing markers of photoreceptors could be accelerated by co-culture with retinal explants, especially when the explants originated from mice bearing a mutation that causes retinal degeneration.T o better characterize the phenotype of retinal cells obtained with this differentiation protocol, a microarray-based analysis comparing human retina to the hESC-derived retinal cells was per-formed (Lamba and Reh 2011).It was demon-strated that gene expression in hESC-derived retinal cells was highly correlated to that in the human fetal retina. In addition, 1% of the genes that were highly expressed in the hESC-derived cultures could be attributed to RPE and ciliary epithelium differentiation.A n alternative protocol for the derivation of retinal progenitors and photoreceptors was pro-posed by Osakada et al. (2008).Similar to the protocol for the derivation of RPE cells, they used serum-free ﬂ oating cultures in combination with the Dkk1 and LeftyA. After 20 days of cul-ture in suspension, the cells were replated on poly-D-lysine/laminin/ﬁ bronectin-coated slides. Osakada and co-authors demonstrated that on day 35 in culture, about 16% of colonies were positive for retinal progenitor markers Rx and Pax6. Differentiation towards photoreceptor fate was augmented in the presence of N2 by treat-ment with retinoic acid and taurine, which are known inducers of rod fate differentiation. Under these conditions, after an extended culture period of 170 days, about 20% of total cells were positive for Crx, an early photoreceptor marker. On day 200, about 8.5% of the cells expressed the mature rod photoreceptor marker, rhodopsin, as well as cone photoreceptor markers, red/green and blue opsins (8.9% and 9.4%, respectively).A n alternative approach was proposed by the same group based on the use of small molecules. In this method, the chemical inhibitors CKI-7 and SB-431542 that inhibit Wnt and Activin A signaling, respectively, and Y-27632, the Rho-associated kinase inhibitor, which prevents disso-ciation-induced cell death, were used. These molecules were shown to mimic the effects of Dkk1 and LeftyA (Osakada et al. 2009).This strategy, which doesn’t involve the use of recom-binant proteins which are produced in animal or E scherichia coli cells, is more favorable for the gen-eration of cells for future transplantation therapy.I n another study that was published by Meyer et al .(2009), after initial differentiation in sus-pension for 6 days, the aggregates were allowed to attach to laminin–coated culture dishes. After further differentiation as adherent cultures, neu-roepithelial rosettes were formed, which were mechanically isolated and subsequently culti-vated as neurospheres. The authors didn’t use any soluble factors; moreover, they showed that under these conditions, the cells expressed endogenous Dkk1 and Noggin. They also demonstrated that in concordance with the role of bFGF in retinal speciﬁ cation, the inhibition of endogenous FGF-signaling abolished retinal differentiation. Under their differentiation protocol, by day 16, more than 95% of the cells expressed the retinal pro-genitor markers, Pax6 and Rx. The authors dem-onstrated that by day 80 of differentiation, about 19% of all neurospheres contained Crx+ cells and within these Crx+ neurospheres, 63% of all cells express Crx and 46.4% of the cells expressed mature markers, such as recoverin and cone opsin.I n all of the above studies, differentiated cells expressing the retinal markers were obtained; however, the cells were not organized in a three-dimensional retinal structure. In a paper recently published by Eiraku et al. (2011),the authors cul-tured free-ﬂ oating aggregates of mouse ES cells in serum-free medium in the presence of base-ment membrane matrix, Matrigel, that could also94M. Idelson and B. Reubinoffbe substituted with a combination of laminin, entactine and Nodal. Using a mouse reporter ES cell line, in which green ﬂ uorescent protein (GFP) is knocked in at the Rx locus, the authors showed that Rx-GFP+ epithelial vesicles were evaginated from the aggregates after 7 days of differentiation under these conditions. On days 8–10, the Rx-GFP+ vesicles changed their shape and formed optic cup-like structures. The inner layer of these structures expressed markers of the neural retina whereas the outer layer expressed markers of RPE. The authors demonstrated that differen-tiation into RPE required the presence of the adjacent neuroectodermal epithelium as a source of diffusible inducing factors. In contrast, the differentiation into neural retina did not require tissue interactions, possibly because of the intrinsic inhibition of the Wnt-signaling pathway. Eiraku and colleagues showed that the retinal architecture, which was formed within the optic vesicle-like structures, was comparable to the native developing neural retina.R ecently, optic vesicle-like structures were also derived from hESCs and iPSCs using the protocol described above, which is based on iso-lating the neural rosette-containing colonies and culturing them in suspension (Meyer et al. 2011). The cells within the structures expressed the markers of retinal progenitors, and after differen-tiation gave rise to different retinal cell types. It was shown that the ability of optic vesicle-like structures to adopt RPE fate could be modulated by Activin A supplementation. The production of these three-dimensional retinal structures opens new avenues for studying retinal development in normal and pathological conditions.T ransplantation of Pluripotent Stem Cell-Derived Retinal CellsA key step towards future clinical transplanta-tions of hESC-derived RPE and neural retina is to show proof of their therapeutic potential i n vivo. Various animal models of retinal degeneration have been used to evaluate the therapeutic effect of transplanted retinal cells. Human ESC-derived RPE cells were transplanted subretinally to the degenerated eyes of RCS rats. Transplantation of the hESC-derived RPE cells between the RPE and the photoreceptor layer rescued retinal struc-ture and function (Lund et al. 2006; Vugler et al. 2008; Idelson et al. 2009; Lu et al. 2009).The subretinally engrafted hESC-derived RPE cells salvaged photoreceptors in proximity to the grafts as was shown by the measurement of the thick-ness of the ONL, the layer of photoreceptor nuclei, which is an important monitor of photore-ceptor cell survival. The ONL thickness was signiﬁcantly increased in transplanted eyes in comparison to the degenerated non-treated eyes.I n order to evaluate the functional effect of transplanted cells i n vivo, the electroretinography (ERG) that directly measures the electrical activ-ity of the outer (a-wave) and inner (b-wave) retina in response to light stimulation was used. It was demonstrated that after transplantation of hESC-derived RPE, ERG recordings revealed a signiﬁ -cant preservation of retinal function in the treated eyes as compared to control untreated eyes (Lund et al. 2006; Idelson et al. 2009).The visual func-tion of the animals was also estimated by an optomotor test, which monitors the animal’s reﬂ exive head movements in response to a rotat-ing drum with ﬁ xed stripes. Animals transplanted with hESC-derived RPE showed signiﬁ cantly better visual performance in comparison to con-trol animals (Lund et al. 2006; Lu et al. 2009). The presence of rhodopsin, a major component of photoreceptor outer segments, within the sub-retinaly transplanted pigmented cells suggested that they could perform phagocytosis i n vivo (Vugler et al. 2008; Idelson et al. 2009).B ridging the gap between basic research and initial clinical trials requires immense resources to ensure safety and efﬁcacy. Human ESC-derived RPE cell lines were generated using a current Good Manufacturing Practices (cGMP)-compliant cellular manufacturing process (Lu et al. 2009). Long-term studies analyzing safety and efﬁcacy of transplantation of these GMP-compliant hESC-derived RPE cells revealed that the subretinally transplanted cells survived for a period of up to 220 days and provided prolonged functional improvement for up to 70 days after transplantation. The potential of the hESC-derived。

视神经损伤后视网膜神经节细胞保护研究进展

・１１４７・・讲座与综述・视神经损伤后视网膜神经节细胞保护研究进展吕燕春１７吕立权２，卢亦成２【中图分类号】Ｒ７７４．６【文献标识码】Ｃ【文章编号】１６７１—０８００（２００９）１０－１１４７．０３外伤、肿瘤、炎疗和缺血均可造成视神经损伤，从而导致患者视力严重受损。

视神经损伤后大量视网膜神经节细胞（ｒｅｔｉｎａｌｇａｎｇｌｉｏｎｃｅｌｌｓ，ＲＧＣｓ）继发死亡，是不町逆视觉功能减退的主要原因。

减缓或抑制视神经损伤后ＲＧＣｓ的继发死亡是有效治疗视神经损伤和促进视觉功能恢复的基础。

最近视神经损伤后ＲＧＣｓ的保护研究取得了一系列进展，提出了许多有临床应用前景的神经保护措施，概括起来包括两大类，即抑制神经元死亡的启动或阻止死亡的执行。

１拮抗毒性物质１．１谷氨酸抑制剂谷氨酸是参与视觉传导的主要的兴奋性递质，研究显示视神经损伤后眼内谷氨酸水平升高，通过ＲＧＣ细胞膜上的Ｎ，甲基．Ｄ．天门冬氨酸盐（ＮＭＤＡ）和ｄ．氨基一３一羟基．５．甲基一４－异唑丙酸盐（ＡＭＰＡ）．红藻氨酸受体导致大最ｃ矿内流、能量耗竭和神经元坏死。

ＮＭＤＡ受体拈抗剂ｍｅｍａｎｔｉｎｅ（美金Ｉ习ＪＪ）、ｄｉｚｏｃｉｌｐｉｎｅ（ＭＫ８０１）和ＡＭＰＡ．红藻氨酸受体拮抗剂ＮＢＱＸ、ＤＢＱＸ均可阻断谷氨酸的兴奋毒作用。

这些物质对成年大鼠、小鼠和灵长类视神经损伤后ＲＧＣｓ均有保护作用“’。

１．２～氧化氮抑制剂视神经横切后，视网膜小胶质细胞和ＭＵｌｌｅｒ自Ｕ胞内诱导型一氧化氮合成酶（ｉｎｄｕｃｉｂｌｅｎｉｔｒｉｃｏｘｉｄｅ基金项目；国家自然科学基金资助项目，编号：３０６００６３５作者单位：１．浙江省余姚市卫生进修学校，浙江余姚３１５４００：２．上海长征医院神经外科．上海２００００３作者简介：吕燕春（１９７４一），女．浙江省余姚市人，主治医师。

ｓｙｎｔｈａｓｅ，ｉＮＯＳ）表达增加，ＲＧＣｓ则诱导表达神经元型ＮＯＳ，给予ＮＯＳ抑制剂可以促进ＲＧＣｓ存活和延缓轴索变性叫。

东洋牌Can Get Signal免疫染色试剂说明书

Instruction manual Can Get Signal immunostain 0810 F0992K፧፧Can Get Signal® immunostain Immunoreaction Enhancer SolutionNKB-401 5 ml x 2NKB-501 20mlNKB-601 20mlStore at 4°C Contents[1] Introduction[2] Components[3] Protocol1. Materials required2. Protocol for paraffin-embedded sections3. Protocol for paraffin-embedded sections using secondary antibody reagentspreviously optimized or for polymer complex method4. Protocol for frozen sections[4] Reagent[5] TroubleshootingTOYO 2CHOME, KOTO-KU, TOKYO, 135-0016, JAPANe-mail:******************.jpTD. TOYOBO Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio ********************FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.[ 1 ] Intro ductio n[ 2 ] Co mpo nentsDescriptionCan Get Signal ® immunostain is a reaction solution that contains an accelerator forantigen-antibody reactions, which improves sensitivity, specificity, and S/N of immunohistochemistry (IHC) and immunocytochemistry.Features-Can Get Signal ® immunostain improves sensitivity, specificity, and S/N of IHC.-This system can be applied to various detection systems (e.g., chromogenic, chemiluminescence, or fluorescence).-This system can be used with ABC or polymer complex methods.-Can Get Signal ® immunostain consists of Solution A and B, which exhibit various properties for improving results. These reagents can be used independently. -Reagents can be used directly without dilution. <Ready-to-use type>Fig. 1 Flow chart of ABC staining with IHCNotes-Can Get Signal ® immunostain cannot be used as a blocking reagent. Blocking and detection steps should be performed using conventional methods.-This reagent is not applicable to the avidin-biotin reaction in ABC methodThis kit includes the following components. All reagents should be stored at 4°C and protected from light.Code No.Reagent Name NKB-401 NKB-501 NKB-601 Solution A 5 ml 20 ml - Solution B5 ml-20 mlNotesCan Get Signal ® immunostain contains Solution A and B. These solutions exhibit various acceleration effects, which are antigen/antibody-dependent, and can be used independently. Both solutions should be examined prior to use.CO., L TD. TOYOBO Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio ********************NOT FOR HUMAN OR DIAGNOSTIC USE.[ 3 ] ProtocolIH C is a method for detection of proteins located in tissue sections, which is accomplished through the use of antibodies that recognize target proteins. The antibody-antigen interaction is visualized by 1) chromogen detection, where an enzyme conjugated to an antibody cleaves a substrate to produce colored precipitate at the protein location, or 2) fluorescent detection, where a fluorophore is conjugated to an antibody and can be visualized using fluorescence microscopy.The following is a protocol for IHC in fixed tissue (typically neutral buffered formalin), which is embedded in paraffin prior to sectioning, using the ABC method with HRP-conjugated antibodies. If secondary antibodies have been previously optimized, or for the polymer complex method (e.g. ENVISION+, Dako), refer to [3] 3.1. Materials required(1) Equipment: - Hellendahl jar - Slide - Coverslip- Mounting medium - Marker pen(2) Reagents and consumables: -Ethanol -Xylene -PBS*-Endogenous peroxidase blocking buffer* -Blocking reagent* -Chromogen substrate2. Protocol for paraffin-embedded sections(1) Place slides in rack and perform the following wash steps: -Xylene: three times for 3 minutes each wash -Ethanol: three times for 3 minutes each wash -90% Ethanol: 3 minutes -80% Ethanol: 3 minutes -70% Ethanol: 3 minutes-Distilled water: 5 minutes-2 hoursNotesFormalin-fixed tissue sections often require an antigen retrieval step prior to IH C staining. During formalin fixation, methylene bridges between proteins are formed and antigenic sites become masked. Several antigen retrieval methods are effective for breaking the methylene bridges and exposing antigenic sites to allow antibodies to bind. H eat-mediated (or heat-induced) or enzymatic antigen retrieval method is generally sufficient.*See [7] ReagentTD. TOYOBO Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio ********************NOT FOR HUMAN OR DIAGNOSTIC USE. (2) Optional : Incubate in endogenous peroxidase blocking buffer for 30 minutes at room temperature in the dark. NotesSome cells or tissues contain endogenous peroxidase. Endogenous peroxidase activity, which may cause high background, can be significantly reduced by pre-treating cells or tissues with hydrogen peroxide prior to incubation with HRP-conjugated antibodies. (3)Optional : Wash in distilled water for 5 minutes, and two times in PBS for 5 minutes.(4) Dilute primary antibodies in Can Get Signal ® Immunostain Solution A or B to an appropriate concentration. NotesCan Get Signal ® Immunostain Solution A and B exhibit different acceleration effects, depending on antigens and antibodies. These solutions can be used independently; however, both solutions should be examined previously.(5) After removing blocking reagent, add 100 P l diluted primary antibody solution and incubate at room temperature for 1 hour. NotesThis reaction can be performed at 4°C overnight. (6) Wash 3 times in PBS for 5 minutes.(7) Dilute secondary antibodies in Can Get Signal ® Immunostain Solution A or B to an appropriate concentration. Notes-Can Get Signal ® Immunostain Solution A and B exhibit different acceleration effects, depending on antigens and antibodies. These solutions can be used independently; however, both solutions should be examined previously.-Optimal antibody concentrations tend to be lower in this method than conventional methods. Therefore, antibody concentrations should be optimized based on lower concentrations.(8) Subsequent to removal of primary antibody solution, add 100 P l diluted secondary antibody solution, and incubate at room temperature for 1 hour. (9) Wash 3 times in PBS for 5 minutes.TD. TOYOBO Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio ********************FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.(10) After removing residual PBS, add 100 P l avidin-biotin complex solution andincubate at room temperature for 30 minutes. NotesThe avidin-biotin complex solution should be used within 30 minutes after preparation. (11) Wash 3 times in PBS for 5 minutes.(12) After removing residual PBS, add 200 P l substrate solution and incubate at room temperature for an appropriate time.(13) Rinse in distilled water to terminate the reaction. (14)Optional : Counterstain.(15) Mount coverslip with aqueous mounting medium or glycerol.3. Protoc ol for paraffin-embedded sec tions using previously optimized secondary antibody concentrations or the polymer complex method(1) Perform step (1)-(6) in [3] 2.(2) After removing residual PBS, add 100 P l secondary antibody solution and incubate at room temperature for 30 minutes. (3) Perform step (9)-(15) in [3] 2.4. Protocol for frozen sections(1) Wash the section 3 times in PBS for 10 minutes.(2) Fix with the pre-cooled fixative (e.g., acetone) for 5-10 minutes at room temperature.(3) Wash in PBS for 10 minutes. (4) Perform (2)-(15) in [3] 2. NOTESThe use of paraffin-embedded sections with previously optimized secondary antibody concentrations, or the polymer complex method ([3] 3.), can be applied to this protocol.TD. TOYOBO Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio ********************NOT FOR HUMAN OR DIAGNOSTIC USE.[ 4 ] Reagents1. 10X PBS(-) (10X PBS) (500 ml)5.75 g Na 2HP04•7H 201.0 g KH 2PO 440.0 g NaCl 1.0 g KClAdjust volume to 500 ml2. Endogenous peroxidase blocking solution (200 ml)194 ml methanol 6 ml 10% H 2O 23. Blocking solution (10 ml)10 ml 1X PBS(-) 150P l normal serumNotesAnimal species should be same between normal serum and secondary antibodies.FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.SymptomCauseSolutionExcessive primary antibodyIn this method, optimal concentrations tend to belower than conventional methods. Therefore, antibody optimization should be based on the lower concentrations.Excessive secondary antibody-In this method, the optimal concentrations for secondary antibodies tend to be lower than conventional methods. Therefore, antibody optimization should be based on lower concentrations.-Previously optimized secondary antibodies can be diluted with this reagent. Insufficient blocking -Prolong blocking time.-Change the blocking reagent. Insufficient washing Increase wash steps or time.Endogenous peroxidase -Prolong treatment time with endogenous peroxidase blocking buffer.-Increase H 2O 2 concentration of endogenous peroxidase blocking buffer up to 3%. High background/ Non-specific signalExcessive exposure time (Fluorescent stain)Decrease exposure time.Insufficient primary antibody Increase concentration of primary antibodies. Excessive blocking - Optimize blocking time. - Change the blocking reagent Excessive washing Decrease wash steps or time.Lack of antigenicity-Tissue fixation method might be inappropriate. Change fixation method.-Antigen retrieval might be effective.Masking of antigenicity Formalin-fixed tissue sections often require antigen retrieval prior to IHC staining. Antigen retrieval method is inappropriateOptimize antigen retrieval conditions. Weak signalExcessive exposure time (Fluorescent stain)Decrease exposure time. Excessive excitation light bleaches fluorescence.[ 5] Tro ublesho o tingTOYO 2CHOME, KOTO-KU, TOKYO, 135-0016, JAPANe-mail:******************.jp Phone : +81-3-5632-9617 FAX : +81-3-5632-9618。

维格列汀对2型糖尿病患者心血管保护作用的研究进展

2020年10月第17卷第20期与非糖尿病患者相比，2型糖尿病（Type 2 Diabetes Mellitus, T2DM）患者罹患心血管疾病（Cardiovascular Diseases, CVDs）的概率是非糖尿病患者的2～6倍，使其成为人口中的主要死亡原因［1-5］。

因此，控制血糖的主要目标应该是预防CVD和微血管疾病造成的死亡和限制发病率［6-8］。

二肽酰肽酶-4（Dipeptidyl Peptidase-4, DPP-4）抑制剂被广泛应用于治疗T2DM。

以下将简述以维格列汀（Vildagliptin）为代表的DPP-4抑制剂对T2DM心血管疾病保护作用的相关研究。

1 维格列汀保护心血管的相关动物试验Furukawa等［9］研究了DPP-4抑制剂维格列汀在压力负荷下对心肌代谢和心脏功能的保护作用。

分别用空白制剂或维格列汀治疗试验小鼠，然后实施横主动脉缩窄术（Transverse Aortic Constriction, TAC）。

TAC术后3周，小鼠心肌肥厚和收缩功能损害显著减轻。

压力-容积分析（Pressure-volume Analysis）显示，维格列汀治疗可明显提高TAC 术后心脏左心室收缩效率。

心肌能量底物分析（Myocardial Energy Substrate Analysis）显示，维格列汀治疗显著增加葡萄糖摄取和脂肪酸摄取。

成纤维细胞生长因子21（Fibroblast Growth Factor 21, FGF21）是一种参与能量代谢调节的多肽，在TAC 术后心脏中表达增强，维格列汀治疗后进一步增强。

维格列汀处理后，FGF21在小鼠心脏成纤维细胞中的表达强于心肌细胞。

维格列汀处理还可通过Sirtuin1（Sirt1）介导的途径诱导人心脏成纤维细胞中FGF21的表达，提示成纤维细胞介导的FGF21在应激心脏中可能调节能量代谢，发挥维格列汀介导的有益作用。

维格列汀通过Sirt1诱导心脏成纤维细胞激发代谢调节因子FGF21的表达，提高了压力负荷下心脏的收缩效率。

211275327_小儿热速清颗粒联合注射用阿莫西林钠舒巴坦钠、维生素C治疗小儿急性化脓性扁桃体炎的

（1）：6799.[4]王磊，沙春洁，余飞，等.醋酸奥曲肽缓释微球在大鼠体内的药动学与药效指标评价研究[J].中国新药杂志，2019，28（11）：1318-1324.[5]赵京平，王誉敏，游云龙，等.艾司奥美拉唑联合阿莫西林方案与含铋剂四联方案对幽门螺杆菌感染初治患者的疗效比较[J].实用临床医药杂志，2022，26（12）：51-55，60.[6]中华医学会外科学分会胰腺外科学组.中国急性胰腺炎诊治指南（2021）[J].中华外科杂志，2021，59（7）：578-587.[7]潘华，叶文冲，戴家超，等.通腑解毒方联合大承气汤保留灌肠和常规治疗对急性胰腺炎患者的临床疗效[J].中成药，2022，44（3）：776-780.[8]吕晓芳，方立峰，陈佩.血清中MIP-1α、MIP-1β水平变化与急性胰腺炎患者A-PACHE Ⅱ评分的关联性及动态监测临床意义[J].实验与检验医学，2021，39（5）：1073-1075.[9]刘春霞，贾秀贤，王利峰，等.穴位贴敷联合芩栀清胰饮治疗急性胰腺炎的疗效观察及对胃肠功能的影响[J].上海针灸杂志，2022，41（7）：656-660.[10]屠冬英，杨丽红，王玲，等.多学科团队协作模式下的延续护理对急性胰腺炎病人健康行为能力和生活质量的影响[J].护理研究，2022，36（2）：317-321.[11] SHEN Y，XUE C，YOU G，et al.miR-9 alleviated theinflammatory response and apoptosis in caerulein-induced acute pancreatitis by regulating FGF10 and the NF-κB signaling pathway[J].Experimental and Therapeutic Medicine，2021，22（2）：795.[12]吴红雪，王厚清，燕宪亮，等.重症急性胰腺炎合并脓毒症与病原菌特征、凝血功能及微循环障碍的相关性研究[J].医学研究杂志，2022，51（3）：108-112.[13]臧珺.醋酸奥曲肽结合大承气汤治疗对急性胰腺炎患者炎症细胞因子水平与肠功能恢复的影响[J].中国实用医药，2021，16（25）：1-4.[14] IWAI N，SAKAGAMI J，KAGAWA K.Gastrointestinal: acutepancreatitis related to a ghrelin receptor agonist[J].Journal of Gastroenterology and Hepatology，2022，37（8）：1473.[15]王以琳，张伟婷，植俊华，等.加味大承气汤治疗急性胰腺炎的效果及对肺功能、炎症因子的影响[J].中外医学研究，2022，20（19）：1-4.[16]肖术平，郑超.艾司奥美拉唑钠改善重症急性胰腺炎患者肠黏膜屏障功能的临床效果研究[J].现代医药卫生，2022，38（18）：3187-3190.[17]孙娜娜.急性胰腺炎住院患者早期胃肠功能与疾病进展的相关性[J].中国实用医药，2021，16（3）：61-63.（收稿日期：2022-11-04）　（本文编辑：冯乐乐）①龙岩市第一医院　福建　龙岩　364000小儿热速清颗粒联合注射用阿莫西林钠舒巴坦钠、维生素C治疗小儿急性化脓性扁桃体炎的效果观察江秀华①　黄丽琴①　林贞①【摘要】　目的：分析小儿热速清颗粒联合注射用阿莫西林钠舒巴坦钠、维生素C 治疗小儿急性化脓性扁桃体炎的效果。

综合标准和代谢信号程序在典型T细胞反应2014.7(33.8)

T cell receptor (TCR) engagement by peptide–MHC complexes initiates a multitude of signalling programmes that prepare the cell for differentiation, proliferation and effector function. The canonical signalling pathways thatlead to activation-induced transcription are mediated by nuclear factor-κB (NF-κB), activator protein 1 (AP-1) and nuclear factor of activated T cells (NFAT). These three pathways collaborate to promote the expression of effector molecules that are crucial for T cell function 1–7 (FIG. 1a). It is generally thought that TCR-induced signalling only leads to T cell activation when it occurs in the context of a second co-stimulatory signal, such as the ligation of CD28full T cell activation 3. Likewise, CD28 signalling leads to the activation of phosphoinositide 3-kinase (PI3K) and the subsequent activation of mammalian target of rapa-mycin (mTOR)9. In addition to co-stimulation, further sig-Recently, the signalling pathways that control cellular metabolism have been shown to have a crucial role in dictating the outcome of T cell activation. Overall, this requirement for the coordination of T cell metabolism and T cell function reflects two important features of the T cell response: the ability of low frequency, antigen-specific naive T cells to rapidly increase in number in response to a pathogen, and their ability to generate long-lived memory T cells or regulatory T (T Reg ) cells that can modulate immune responses. In this Review, we aim to integrate the metabolic pathways with the canoni-cal T cell signalling pathways to provide a comprehen-sive view of the pathways that regulate T cell immunity. This reveals potential new pharmacological targets for enhancing or inhibiting specific T cell responses.Regulation of cellular metabolismCellular metabolism provides the means by which cells store and use macromolecules that are necessary for growth and for the generation of energy. Depending on nutrient availability and external or intracellular cues, Sidney KimmelComprehensive Cancer REVIEWS|Immunology bGlucose transporter type 1 (GLUT1). A unidirectional transporter that facilitates the transport of glucose across the plasma membrane.Lactate dehydrogenase A (LDHA). An enzyme that catalyses the conversion of pyruvate to lactate.Hexokinase 2An enzyme that initiates the first reaction of glycolysis by phosphorylating glucose to produce glucose‑6‑phosphate.tricarboxylic acid cycle (TCA cycle) and promote theoxidative phosphorylation of energy inter m ediatesin the mitochondrial matrix to generate a total of~30 ATP molecules (TABLE 1). If oxygen is unavailable,the two molecules of pyruvate that are generated fromglyco l ysis can be converted to lactate, which dramaticallyreduces the ATP yield but still provides an energy sourcefor the cell10. In response to environmental cues, thereare specific drivers of cellular metabolism that regulatethe expression of enzymes that are crucial for variousmetabolic processes.Glycolysis is promoted by the upregulation of MYC,which is a basic helix–loop–helix leucine zipper tran-scription factor (TABLE 2). MYC promotes the expres-sion of glucose transporter type 1 (GLUT1; also knownas SLC2A1), pyruvate kinase, lactate dehydrogenase A(LDHA) and hexokinase 2, which are required for glu-cose uptake and for the rate-limiting steps of glyco-lysis11,12. In addition, MYC promotes the expression ofboth glutaminase and glutamine transporters13, andfurther promotes glutaminolysis by transcriptionallyrepressing the microRNAs miR-23a and miR-23b, whichallows for the increased expression of glutaminase14.Furthermore, MYC has also been found to have a rolein promoting mitochondrial biogenesis15.Glycolysis is also regulated by hypoxia-induciblefactor 1α (HIF1α), which is a heterodimeric basichelix–loop–helix and Per–Arnt–Sim (PAS) domain-containing transcription factor that, during hypoxia,binds to cis-acting hypoxia-response elements and leads engagement) in the setting of signal 2 (co-stimulation; depicted as CD28) leads to full T cell activation122. This is facilitated by the activation of three canonical transcription factors — nuclear factor-κB (NF-κB), activator protein 1 (AP-1) and nuclear factor of activated T cells (NFAT)6,7,123,124. This, in turn, leads to the expression of multiple cytokines, chemokines and cell surface receptors, all of which promote T cell activation and proliferation3. Alternatively, TCR recognition alone (in the absence of co-stimulation) leads to an ‘off’ signal in the form of T cell anergy5,125. Under these conditions, NFAT is activated in the absence of full AP-1 activation, which leads to the expression of genes such as diacylglycerol kinase-α (DGKA) and the E3 ubiquitin-protein ligases CBLB and GRAIL (which encodes gene related to anergy in lymphocytes; also known as RNF128), which inhibit full T cell activation3. b | Upon T cell activation, cytokines in the T cell microenvironment determine the outcome of antigen recognition with regard to effector T cell differentiation51. As shown for CD4+ T cells, interleukin-12 (IL-12), IL-4 and IL-6 activate signal transducer and activator of transcription 4 (STAT4), STAT6 and STAT3, respectively. This leads to the expression of T-bet, GATA-binding protein 3 (GATA3) and retinoic acid receptor-related orphan receptor-γt (RORγt), which facilitates the generation of T helper 1 (TH1), TH2 and TH17 cells. Alternatively, transforming growth factor-β (TGFβ) signalling through SMAD2–SMAD4 promotes the expression of forkhead box P3 (FOXP3) and the generation of regulatory T (TReg) cells.DAG, diacylglycerol; IKK, inhibitor of NF-κB kinase; InsP3, inositol-1,4,5-trisphosphate; LAT, linker for activation ofT cells; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; PI3K, phosphoinositide 3-kinase; PKCθ, protein kinase Cθ; PLCγ, phospholipase Cγ; PtdInsP2, phosphatidylinositol-4,5-bisphosphate; SLP76, SH2 domain-containing leukocyte protein of 76 kDa (also known as LCP2); ZAP70, ζ-chain-associated protein kinase of 70 kDa.Pyruvate dehydrogenase kinase 1(PDK1). An enzyme that phosphorylates and inactivates pyruvate dehydrogenase, thereby inhibiting the catalysis of pyruvate to acetyl‑CoA and preventing the initiation of the tricarboxylic acid cycle. Phosphofructokinase 1A rate‑limiting enzyme of glycolysis that requires ATP to convert fructose‑6‑phosphate into fructose‑1,6‑bisphosphate. Carnitine palmitoyltransferase 1A(CPT1A). A rate‑limiting mitochondrial enzyme that is necessary for fatty acid oxidation. CPT1A catalyses the transfer of the acyl group of long‑chain fatty acids to acylcarnitine, which allows for its transport from the cytosol to the mitochondria.to the transcription of numerous genes that are impor-tant for cell survival in low oxygen conditions16(TABLE 2).Not surprisingly, these genes include those encodingenzymes that are required for the glycolytic pathway17.In addition, HIF1α promotes the expression of GLUT1(REF. 18) and enforces ATP synthesis by glycolysis, ratherthan oxidative phosphorylation, by upregulating pyruvatedehydrogenase kinase 1 (PDK1), which is an enzyme thatinhibits the entry of pyruvate into the TCA cycle19,20.HIF1α expression is not only regulated by oxygenlevels but also depends on external cues that are inte-grated by mTOR activity21. mTOR is an evolutionar-ily conserved serine/threonine kinase that integratesa diverse array of environmental cues to regulategrowth, survival and proliferation22(TABLE 2). mTORis present in two distinct protein complexes — mTORcomplex 1 (mTORC1) and mTORC2 — that each haveunique downstream targets and functions. Activation ofmTORC1 occurs by growth factor stimulation of PI3K,which initiates a signalling cascade that results in theinhibitory phosphorylation of the mTORC1 repressortuberous sclerosis 2 (TSC2; also known as tuberin) bythe kinase AKT23. In addition to growth factors, aminoacids also activate mTORC1 and this leads to recruit-ment of mTOR to the lysosomal surface where it caninteract with, and become activated by, its activatorRAS homologue enriched in brain (RHEB)24–26. Themechanisms that regulate mTORC2 activation are lessclear than those for mTORC1. However, it is known thatgrowth factor stimulation enhances mTORC2 activityand recent studies have implicated a role for the asso-ciation of the mTORC2 complex with ribosomes inpromoting its activation27.The activity of mTORC1 enhances HIF1α expressionat both the transcriptional and translational level, andthereby stimulates glycolysis and glucose transport28. Theimportance of HIF1α in mediating mTORC1-enhancedglycolysis is illustrated by the observation that smallinterfering RNA (siRNA)-mediated inhibition of Hif1aexpression in cells that express constitutively activemTORC1 (Tsc2−/− cells) abrogates the expression of theglycolytic factors GLUT1, phosphofructokinase 1 andPDK1 (REF. 28). Interestingly, a recent report suggeststhat MYC activity is, in part, regulated by mTORC2(REF. 29). It was observed that mTORC2 activity leads tothe acetylation of forkhead box protein O1 (FOXO1),which initiates the release of MYC from a suppressivemiR-34c-dependent network29.Although MYC, HIF1α and mTOR signalling pro-mote an increased metabolic output by cells, other reg-ulators promote energy conservation during times oflimited resources. One such regulator is AMP-activatedprotein kinase (AMPK), which is a heterotrimericserine/threonine kinase complex that monitors cellularenergy levels (TABLE 2). The binding of AMP or ADP toAMPK induces its phosphorylation and activation byupstream kinases30,31. AMPK activation enhances glucoseuptake and, at the same time, inhibits glucose, glycogen andfatty acid synthesis31. This occurs through the phosphoryl-ation and inhibition of acetyl-CoA carboxylase 1 (ACC1)and the inhibition of the lipogenic transcription factorsterol regulatory element-binding protein 1 (SREBP1;also known as SREBF1)32. In addition, AMPK promotesfatty acid oxidation through the phosphorylation andinhibition of ACC2. This results in the enhanced expres-sion of carnitine palmitoyltransferase 1A (CPT1A), which isTable 1 |A summary of metabolic pathways and moleculestransferase 1A; GLUT, glucose transporter; PDK1, pyruvate dehydrogenase kinase 1; SREBPs, sterol regulatory element-binding proteins; TCA cycle, tricarboxylic acid cycle.REVIEWSAutophagyAn evolutionarily conserved process in which acidic double‑membrane‑bound vacuoles sequester intracellular contents (such as damaged organelles and macromolecules) and target them for degradation through fusion with secondary lysosomes.αketoglutarateA key intermediate of the tricarboxylic acid cyclethat can be derived from glutaminolysis.the rate-limiting factor in mitochondrial lipid uptake10.AMPK also enhances mitochondrial bio g enesis andoxidative metabolism by promoting the transcrip-tional activity of peroxisome proliferator-activatedreceptor-γ co-activator 1α (PGC1α; also known asPPARGC1A)31. Thus, AMPK regulates cell metabolism tolimit energy expenditure and replenish ATP production.AMPK activity can also diminish mTORC1 signallingthrough the phosphorylation of TSC2 and regulatory-associated protein of mTOR (RAPTOR; also knownas RPTOR), which is a crucial component of mTORC1(REFS 33,34). Under conditions of prolonged energy depri-vation (starvation), AMPK promotes autophagy by phos-phorylating and activating the serine/threonine proteinkinase Unc-51-like kinase 1 (ULK1)35. Thus, AMPKshuts down energy-demanding synthetic pathwaysbut promotes mechanisms that generate energy —such as glycolysis, oxidative phosphorylation andautophagy — as a means of deriving substrates fromwithin the cell. By contrast, deficiency of the AMPKactivator liver kinase 1 (LKB1; also known as STK11),and therefore loss of AMPK activation, promotesenhanced glucose and glutamine metabolism throughthe mTORC1-dependent upregulation of HIF1α duringnormoxic conditions36.Interestingly, although mTORC1 activity hasbeen shown to increase glycolysis through regula-tion of HIF1α, mTORC1 activity can also promoteoxidative phosphorylation. This occurs through theincreased interaction of the transcriptional repres-sor yin and yang 1 (YY1) with PGC1α, which inducesthe expression of mitochondrial genes37. In addition,mTORC1 promotes lipid biosynthesis by enhancingthe transcription and translation of SREBP1 (REF. 28)but limits fatty acid oxidation through the inhibitionof CPT1A38. The activity of mTORC1 has also beenshown to promote nucleotide synthesis. This processoccurs through the activation of ribosomal protein S6kinase β1, which post-translationally regulates de novopyrimidine synthesis39,40.T cells have a specialized metabolismMost cells use oxidative phosphorylation to maximizeATP production but activated T cells (and cancer cells)mainly generate ATP through glycolysis41,42. This use ofglycolysis in the presence of oxygen was first described byOtto Warburg for cancer cells and it is therefore referredto as the Warburg effect43. Although glycolysis providesless ATP than oxidative phosphorylation, it has beenproposed that avoiding oxidative phosphorylation allowsfor the generation of substrates that are required for thesynthesis of amino acids, nucleic acids and lipids, all ofwhich are vital for proliferation44. Of note, a recent reporthas challenged the necessity of Warburg physiology inT cell proliferation, instead suggesting that glycolysis isrequired to release translational inhibition of the mRNAthat encodes the effector cytokine interferon-γ (IFNγ)45.Nonetheless, glucose uptake is essential for glyco l ysis andenhanced cell surface expression of GLUT1 is a crucialaspect of TCR-induced T cell activation46. In this regard,it has been shown that CD28 signalling upregulatesthe expression of glucose transporters47. Similarly, theuptake and metabolism of the amino acid glutamine isessential for T cell activation, as glutamine deprivationblocks T cell proliferation and cytokine production11,48.Glutamine oxidation can lead to the generation ofα‑ketoglutarate, which is a key intermediate of the TCAcycle and which, in turn, provides substrates for thegeneration of various macromolecules10. Furthermore,T cells require fatty acid metabolism for their prolifera-tion and function. Cholesterol synthesis is essential formembrane biogenesis and T cells that are deficient inSREBPs (owing to a T cell-specific deletion of SREBPcleavage-activating protein (SCAP)) have diminishedproliferative capacity and reduced antiviral responses49.Along these lines, the expression of SREBP1 is enhancedfollowing TCR activation through the suppression ofthe liver X receptor signalling pathway50. Thus, T cellactivation induces metabolic changes that allow forprocesses that are necessary to promote proliferationand cytokine secretion.receptor-related orphan receptor-γt; TCR, T cell receptor; TH cell, T helper cell; TRegcell, regulatory T cell.Although the considerations that are described above reflect the metabolic needs of T cells during activation and robust proliferation, it has become clear that differ-ent T cell subsets have unique metabolic needs. Thus, T cell activation and fate are linked to specific metabolic programmes that support selective T cell functions. In this regard, immunological signals such as co-stimulatory ligands, cytokines and antigen promote metabolism. Likewise, metabolic signals such as nutrient availa b ility, hypoxia and growth factors regulate immune func-tion. From a signalling perspective, central roles for the energy sensor AMPK and the evolutionarily conserved PI3K family member mTOR in integrating immuno-logical and metabolic pathways have emerged (TABLE 2). Similarly, the transcription factors MYC and HIF1α are central to promoting the expression of the genes that are required for metabolic programmes that support T cell responses (TABLE 2). Focusing on the metabolic and immunological roles of these molecules provides an integrative picture of the signalling pathways that regulate T cell activation, differentiation and function. In the following sections, we discuss the roles of these metabolic factors in influencing CD4+ and CD8+ effec-tor T cell differentiation (FIG. 2), CD8+ memory T cellgeneration and CD4+T Reg cell function (FIG. 3).Metabolic regulation of CD4+ T H cell lineages CD4+ T cells differentiate into distinct helper cell lin-eages following TCR engagement and cytokine stimu-lation 51 (FIG. 1b). To determine whether T H cell subsets have distinct metabolic needs, the metabolic profiles of stimulated T H 1, T H 2 and T H 17 cells have been assessed 52. This study showed that all CD4+ T H cell subsets upregu-late GLUT1 expression upon TCR activation and have elevated glycolytic rates 52. Thus, T H 1, T H 2 and T H 17 cells all use glycolysis upon activation.Recent studies have elucidated a role for MYC in establishing the metabolic profile that is required for effective T cell proliferation. T cell activation induces protein-level expression of both MYC and HIF1α within 2 hours of stimulation 11, and MYC expres-sion levels are highest in proliferating lymphocytes 53. However, MYC — but not HIF1α — is required for upregulating the expression of the glycolytic machin-ery and the substrates that are essential for glutamine metabolism. Deletion of Myc abrogates the ability of activated T cells to undergo glycolysis and to initiate the catabolism of glutamine 11. Furthermore, MYC deficiency diminishes the expression of the glu-tamine exchanger CD98 (a heterodimer of SLC3A2 and SLC7A5), which reduces mTORC1 activity. The absence of MYC in T cells markedly inhibits activation-induced glutaminolysis, and the subsequent generation of nucleotides and polyamines that is necessary for proliferation 11.Although HIF1α is not required for CD4+ T cell proliferation or interleukin-2 (IL-2) production 11,54, several groups have shown that is has an important role in the generation and function of T H 17 cells 54,55. HIF1α expression is highly induced under T H 17 cell-polarizing conditions during T cell activation. This upregulationof HIF1α expression is dependent on signal trans-ducer and activator of transcription 3 (STAT3) and, importantly, occurs even under normoxic conditions 55. Furthermore, HIF1α promotes T H 17 cell differentiation by directly inducing the transcription of the gene that encodes retinoic acid receptor-related orphan receptor-γt (RORγt), and by cooperating with RORγt and the histone acetyltransferase p300 (also known as EP300) to drive the transcription of T H 17 cell-associated genes 55. In addi-tion, it was found that polarizing T cells in vitro under conditions of 5% oxygen promotes T H 17 cell differen-tiation in an mTORC1–HIF1α-dependent manner 56. Under T H 17 cell-polarizing conditions, HIF1α pro-motes the transcription of the genes encoding the rate-limiting enzymes of glycolysis, such as hexo k inase 2, glucose-6-phosphate isomerase, pyruvate kinase and LDHA, as well as GLUT1 (REF . 54).HIF1α expression has also been linked to the main-tenance of T H 17 cells 57. By studying T cells from patients with inflammation, it was observed that T H 17 cells resemble long-lived effector memory cells 57. Indeed, HIF1α was shown to have an important role in maintain-ing the expression of high levels of anti-apoptotic genes in T H 17 cells 57. In addition to HIF1α, the maintenance of T H 17 cells has been linked to the upregulation of T cell factor 7 (TCF7; also known as TCF1) and lymphoid enhancer-binding factor 1 (LEF1), which are targets of the WNT–β-catenin pathway that are expressed at high levels in stem cells 58. Interestingly, work that has been carried out in neural stem cells has shown that HIF1α positively regulates the expression of TCF7 and LEF1 (REF . 59). Although further work will be necessary to sup-port a role for HIF1α in promoting the expression of TCF7 and LEF1 in lymphocytes, these data suggest that HIF1α expression may induce stem cell-like properties in T H 17 cells. Thus, HIF1α coordinates immunological programmes — such as RORγt expression and fork-head box P3 (FOXP3) degradation (see below) — with metabolic programmes (for example, the upregulation of the glycolytic machinery and inhibitors of apoptosis) to promote the development of T H 17 cells.Of note, one group has shown increased T cell activa-tion and IFNγ production in T cells that are deficient for the alternatively spliced isoform of HIF1α known as I.1 (REF . 60). As IL-17 has been shown to inhibit IFNγ pro-duction, it has been proposed that the increase in IFNγ in these mice is due to decreased IL-17 production 55. Nonetheless, follow-up studies have revealed that deletion of the HIF1α isoform I.1 in T cells enhances immunity in a model of bacterial infection 61. Therefore, the precise role of HIF1α in T H 1 and T H 2 cell differentiation and function remains to be determined.Dissecting the mTOR pathway has revealed a cru-cial role for mTOR in the regulation of CD4+ T cell lineage differentiation. T cell-specific deletion of Mtor results in the abrogation of T H 1, T H 2 and T H 17 cell dif-ferentiation 62. Instead, stimulation of mTOR-deficient CD4+ T cells induces the accumulation of FOXP3+ T Reg cells 62. Furthermore, a specific deletion of Rheb (leading to the loss of mTORC1) in T cells results in the loss of T H 1 and T H 17 cell differentiation, althoughREVIEWS| ImmunologyT H 2 cell generation is unaffected 63. By contrast, T cells that lack rapamycin-insensitive companion of mTOR (RICTOR), and thus lack mTORC2, are readily skewed towards T H 1 or T H 17 cell lineages (depending on which Cre recombinase is used) but they fail to differenti-ate into T H 2 cells 63,64. In addition, RICTOR-deficient mice are resistant to T H 2 cell-mediated diseases 63,65. Thus, mTORC1 is required for T H 1 and T H 17 celldifferentiation, and mTORC2 is necessary for T H 2 cell development. Although the metabolic profiling of these cells is currently an area of active investigation, we propose that mTOR regulates the metabolic poten-tial of these cells to influence T cell differentiation. Of note, recent papers demonstrate that deletion of the mTORC1 component RAPTOR prevents the genera-tion of T H 1, T H 2, T H 17 and T Reg cells 66,67. This is differentFigure 2 | Integrating immunological and metabolic signalling programmes to promote effector T cell generation and function. The figure shows the coordinated integration of canonical T cell signalling (blue) and metabolic regulators (green) to promote the generation and function of effector T cells. In this perspective, hypoxia-inducible factor 1α (HIF1α) and MYC are just as integral to T cell effector generation as nuclear factor of activated T cells (NFAT), activator protein 1 (AP-1) and nuclear factor-κB (NF-κB). Similarly, mammalian target of rapamycin (mTOR) signalling is as crucial in effector T cell activation and differentiation as the activation of mitogen-activated protein kinase (MAPK), protein kinase C θ (PKC θ) and calcineurin. Thicker arrows indicate the activation of metabolic programmes. Thinner arrows indicate signalling cascades. DAG, diacylglycerol; FOXP3, forkhead box P3; GLUT, glucose transporter;IKK, inhibitor of NF-κB kinase; IL-6R, interleukin-6 receptor; InsP 3, inositol-1,4,5-trisphosphate; LAT, linker for activation of T cells; MAPKK, MAPK kinase; PI3K, phosphoinositide 3-kinase; PKC θ, protein kinase C θ; PLC γ, phospholipase C γ; PtdInsP 2, phosphatidylinositol-4,5-bisphosphate; SLP76, SH2 domain-containing leukocyte protein of 76 kDa (also known as LCP2); STAT, signal transducer and activator of transcription; TCR, T cell receptor; T H cell, T helper cell; ZAP70, ζ-chain-associated protein kinase of 70 kDa.| ImmunologyCD4 or from Rheb −/− mice, in which there are only defects in T H 1 and T H 17 cell differentiation 63. Thus, RHEB-dependent mTORC1 signalling seems to have more selective effects on immune cells. We speculate that the differences that have been observed between Raptor −/− T cells and Rheb −/− T cells are due to markedly enhanced or unopposed mTORC2 activity in Raptor −/− T cells, as many of the defects that are seen in these mice are not observed in mTOR-deficient T cells 62. Nonetheless, the differences between the Raptor −/− and Rheb −/− T cells provide an opportunity to define mTORC1-dependent processes that selectively regulate T cell differentiation.It should be pointed out that, in contrast to these two studies 63,64 on the role of RHEB and RAPTOR in T cells, there is a report suggesting that RAPTOR (and therefore mTORC1) is not required for T H 1 and T H 2 cell differen-tiation and is only crucial for T H 17 cell differentiation by enhancing the nuclear accumulation of RORγt 68. An explanation for these discrepant findings remains to be elucidated. We speculate that the inconsistent results may be related to the enhanced expansion of a CD4−IFNγ+ cell population that can rapidly proliferate in cell cul-tures after magnetic isolation of RAPTOR-deficient CD4+ T cells (J.D.P ., unpublished observations).Figure 3 | Integrating immunological and metabolic signalling programmes to promote CD8+CD4+ regulatory T cell generation. This figure depicts the integration of the canonical T cell signalling pathways(blue) and metabolic regulators (green for activated and red for inhibited). AMP-activated protein kinase (AMPK) activation promotes metabolic programmes that enhance the generation of memory and regulatory T (T Reg ) cells. Alternatively, it is the inhibition of mammalian target of rapamycin (mTOR) and hypoxia-inducible factor 1α (HIF1α) activation that promotes the generation of CD8+ memory or CD4+ regulatory T Reg cells. From this perspective, memory T cells and T Reg cells share similar metabolic requirements. Thicker arrows indicate the downstream consequences of AMPK activation, and of the inhibition of mTOR and HIF1α. AP-1, activator protein 1; DAG, diacylglycerol; FOXP3, forkhead box P3; IKK, inhibitor of NF-κB kinase; InsP 3, inositol-1,4,5-trisphosphate; LAT, linker for activation of T cells; LKB1, liver kinase B1; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase; PKC θ, protein kinase C θ; PLC γ, phospholipase C γ; PtdInsP 2, phosphatidylinositol-4,5-bisphosphate; SLP76, SH2 domain-containing leukocyte protein of 76 kDa (also known as LCP2); TCR, T cell receptor; T H cell, T helper cell; ZAP70, ζ-chain-associated protein kinase of 70 kDa.REVIEWSOther studies have used LKB1-deficient CD4+ T cells (which display a loss of AMPK activation) to investigate the roles of the AMPK and mTOR path-ways in T H cell differentiation. LKB1-deficient CD4+ T cells show elevated production of IFNγ and IL-17, and have an enhanced propensity to differentiate into T H 1 or T H 17 cells 69. LKB1 deficiency also results in enhanced glucose uptake with elevated protein-level expression of GLUT1 and hexokinase 2, which indi-cates that AMPK activation represses glycolysis 69. In addition, LKB1-deficient T cells have increased expres-sion of mTORC1 gene targets compared with wild-type cells, which further supports a crucial role for mTOR in T H 1 and T H 17 cell differentiation 69.The metabolic link to CD8+ effector T cell function CD8+ effector T cells rely heavily on glycolysis to sup-port their metabolic needs during their rapid prolifera-tion in response to infection 70. Inhibition of glycolysis during the activation of naive CD8+ T cells abrogates effector cell generation 71. For example, the glucose analogue 2-deoxy-d-glucose inhibits glycolysis and downregulates the expression of mRNAs encoding the CD8+ T cell effector proteins IFNγ and perforin 72,73. Interestingly, IL-2 production is unperturbed by 2-deoxy-d-glucose treatment.MYC has been shown to be crucial for T cell pro-liferation and its requirement in T cell activation has been further highlighted by studies examining CD8+ T cell function in Myc +/− mice. CD8+ T cells that lack one copy of the Myc gene show impaired activation, as determined by the abrogated upregulation of CD44 (REF . 74). In addition, studies of HIF1β-deficient T cells have provided an insight into the role of HIF1β in CD8+ T cell effector differentiation and function 75. Upon initial activation, HIF1β-deficient T cells read-ily take up glucose and initiate glycolysis. This is in contrast to MYC-deficient CD8+ T cells, which lack the ability to initiate activation and glycolysis 11. However, in response to IL-2, HIF1β-deficient CD8+ T cells fail to sustain GLUT1 levels and they have reduced expression of the key rate-limiting glyco-lytic enzymes, such as hexokinase 2, pyruvate kinase, phospho f ructokinase 1 and LDHA 75. Concomitantly, the HIF1β-deficient CD8+ T cells have reduced expres-sion of effector molecules (namely, perforin and gran-zymes), but proliferation, IFNγ production and T-bet expression remain intact. Of note, these data indicate that the induction of HIF1β depends on mTORC1 activity and thus, mTOR is a crucial regulator of the glycolytic machinery that is upregulated by HIF1β expression 75. Interestingly, this study also showed that mTOR activation in cytotoxic T lymphocytes (CTLs) occurs independently of AKT activation. This suggests that CTLs may use an alternative signalling pathway for the activation of mTORC1 (REF . 75).Consistent with this study is a recent report that examines the function of Von Hippel–Lindau disease tumour suppressor (VHL)-deficient CD8+ T cells, which have enhanced expression of HIF1α76. Compared with wild-type T cells, VHL-deficient T cells have enhancedeffector activity and they more potently reject tumours. Interestingly, although such cells expressed increased levels of effector molecules — such as perforin and granzymes — the overexpression of HIF1α also resulted in the increased expression of inhibitory molecules, such as cytotoxic T lymphocyte antigen 4 (CTLA4) and lymphocyte activation gene 3 protein (LAG3)76.In addition, branched chain amino acids activate the mTOR pathway, as well as providing the building blocks for protein synthesis. A recently defined feature of T cell activation is the increased cell surface expression of the neutral amino acids transporter solute carrier family 7 member 5 (SLC7A5; also known as LAT1). Deletion of SLC7A5 in T cells markedly inhibits clonal expansion and effector cell differentiation 77. Similarly, T cell-specific deletion of Raptor abrogates CD8+ T cell effector func-tion (including IFNγ production) and proliferation in response to infection 66. Furthermore, RAPTOR deficiency results in the downregulation of glycolytic transcripts and MYC protein, and the generation of transcripts that are important in lipid synthesis and oxidative phosphoryla-tion 66. Thus, the RAPTOR–mTORC1 pathway coordi-nates metabolic programmes that are important for T cell activation and function.AMPK is activated by an increase in the AMP/ATP ratio, as well as following TCR engagement. Interestingly, the activation of AMPK following antigen recogni-tion requires the activation of calcium/calmodulin- dependent protein kinase kinases (CaMKKs) but this is not necessary for the activation of AMPK by an increase in the AMP/ATP ratio. These results suggest that in lymphocytes, AMPK activation in response to antigen anticipates ATP depletion even in the pres-ence of adequate nutrients 78. Nonetheless, CD8+ T cells that lack expression of the catalytic α1-subunit of AMPK (AMPKα1) are activated, proliferate and secrete cytokines to an extent that is similar to wild-type T cells 79,80. Thus, AMPK activation is dispensable for T cell activation in the presence of adequate nutri-ents. However, metabolic stress due to glucose depriva-tion induces enhanced cell death in AMPKα1-deficient T cells 80. Similarly, T cells that are deficient in tuber-ous sclerosis 1 protein homologue (TSC1; also known as hamartin) have increased mTOR activation and show increased apoptosis as a result of abnormal mito-chondrial potential and the increased production of reactive oxygen species 81–83.Memory and T Reg cells are metabolically alikeIt has been established that glucose uptake and a high glyco l ytic rate are required for effective CD4+ and CD8+ T cell responses, but both peripherally derivedT Reg cells and CD8+memory T cells do not primarily use glycolysis for energy generation and instead rely on fatty acid metabolism 52,84. Compared with effector T cells,CD8+memory T cells have enhanced mitochondrial spare respiratory capacity, which provides the extra energy stor-age that is necessary to promote survival 85. Memory T cells must also respond rapidly following antigen re c hallenge. In this regard, it was found that memory T cells have a greater mitochondrial mass compared with naiveREVIEWS。

成体神经发育adult nerogenesis

Duan et al, Current Opinion in Neurobiology 2008, 18:1–8
Developmental Process of Newborn Neurons in Hippocampus
(labeled by GFP retrovirus)
Zhao et al, J. Neurosci., January 4, 2006 • 26(1):3
More tasks need to be applied.
Adult neurogenesis in rodents
• Olfactory bulb and hippocampus
Zhao et.al. Cell 132, 645–660, February 22, 2008
Neurogenesis in SVZ
Zhao et.al. Cell 132, 645–660, February 22, 2008
Environmental Regulation of Adult Neurogenesis • Physiological stimuli • Pathological states
Exercise and enriched environment increase adult hippocampal neurogenesis
• Antidepressants increase SGZ neurogenesis
Czeh et al ,PNAS October 23, 2001
Adult neurogenesis is necessary for the effects of antidepressants
S, sham; X, x-ray; V, vehicle; F, fluoxetine

biol reprod

BIOLOGY OF REPRODUCTION(2013)89(4):78,1–11Published online before print14August2013.DOI10.1095/biolreprod.113.110957A MicroRNA(mmu-miR-124)Prevents Sox9Expression in Developing Mouse Ovarian Cells1Francisca M.Real,3Ryohei Sekido,4Darı´o G.Lupia´n˜ez,3Robin Lovell-Badge,4Rafael Jime´nez,2,3and Miguel Burgos33Departamento de Gene´tica e Instituto de Biotecnologı´a,Universidad de Granada,Centro de Investigacio´n Biome´dica, Granada,Spain4Division of Stem Cell Biology and Developmental Genetics,Medical Research Council National Institute for Medical Research,Mill Hill,London,United KingdomABSTRACTIn mammals,sex differentiation depends on gonad develop-ment,which is controlled by two groups of sex-determining genes that promote one gonadal sex and antagonize the oppositeone.SOX9plays a key role during testis development in all studied vertebrates,whereas it is kept inactive in the XX gonad at the critical time of sex determination,otherwise,ovary-to-testis gonadal sex reversal occurs.However,molecular mech-anisms underlying repression of Sox9at the beginning of ovarian development,as well as other important aspects of gonad organogenesis,remain largely unknown.Because there is indirect evidence that micro-RNAs(miRNA)are necessary for testicular function,the possible involvement of miRNAs in mammalian sex determination deserved further ing microarray technology,we have identified22miRNAs showing sex-specific expression in the developing gonads during the critical period of sex determination.Bioinformatics analyses led to the identification of miR-124as the candidate gene for ovarian development.We knocked down or overexpressed miR-124in primary gonadal cell cultures and observed that miR-124 is sufficient to induce the repression of both SOX9translation and transcription in ovarian cells.Our results provide the first evidence of the involvement of a miRNA in the regulation of the gene controlling gonad development and sex determination.The miRNA microarray data reported here will help promote further research in this field,to unravel the role of other miRNAs in the genetic control of mammalian sex determination.cell transfection,gene expression,gonad development, microarray,microRNA,miR-124,ovary,regulation of translation, sex determinationINTRODUCTIONThe process of sex determination involves mechanisms that prompt the undifferentiated embryonic gonad to follow either of the two alternative developmental pathways,the testis or the ovary.A single gene located on the Y chromosome,the sex-determining region of the Y(SRY)gene,is the switch for sex determination in almost all mammals[1–3].In XY mice, shortly after the expression of Sry in somatic cells of the supporting cell lineage of the gonad,testis development begins with the differentiation of these cells as pre-Sertoli cells located in the testis cords that also enclose the primordial germ cells. Several additional cellular events also occur outside the testis cords,including mesonephric cell migration,testis-specific vascularization,and differentiation of Leydig and peritubular myoid cells[see refs.4and5for reviews].In females,the genetic pathways for ovary development are largely unknown to date.Many genes are currently known to be involved in gonadal development[6–8].Expression of SRY-related HMG-box, gene9(SOX9)is essential for normal testis development in most vertebrates.Ectopic expression of Sox9in the gonads of XX mice leads to a female-to-male sex reversal with the formation of XX testes[9,10],and XY mice lacking Sox9 expression develop ovaries,thus showing a male-to-female sex reversal[11,12].Hence,Sox9,like Sry,is necessary and sufficient to trigger testis organogenesis.A positive feedback loop between Sox9and fibroblast growth factor9(Fgf9)is initiated by Sry,which results in the up-regulation of Fgf9and the repression of wingless-type MMTV integration site family, member4(Wnt4)in the male gonad[13].This is crucial for the maintenance of testis development,because Wnt4represses the migration of endothelial and steroidogenic cells from the mesonephros to the XX gonad[14],probably by activating the b-catenin signaling pathway.The fact that XY Fgf9/Wnt4 double mutants developed testes indicates that the primary role of Fgf9is the repression of Wnt4and suggests that the Sox9/ Fgf9positive feedback loop is established through Wnt4,thus Fgf9repressing a Sox9repressor[15].Ovarian differentiation was assumed to be the default pathway in gonad development.However,several discoveries have shown that the ovarian phenotype,once determined,must be actively maintained throughout life.Although Foxl2is required to maintain the ovarian phenotype at the postnatal stage[16],little is known on how ovary differentiation is initiated at the embryonic stage after sex determination.Sex determination is established in terms of a balance between Fgf9-and Wnt4-expression predominance.It is known that Sry tips the balance toward the testis pathway in males,and Wnt signaling may have a similar role in females.R-spondin family member1(RSPO1),which,like Wnt4,serves to stabilize b-catenin,appears sufficient to block the testicular pathway in some XX humans[17],although this is not the case in mice, where XX Rspo1null mutant gonads have a phenotype similar1Supported by the Andalussian Gobernment,Junta de Andalucı´a, through grants CVI-2057and BIO-109,by the Spanish Ministry of Science and Innovation through grant CGL2011-23368,and by the UK Medical Research Council(U117512772).2Correspondence:Rafael Jime´nez,Universidad de Granada,Genetica Facultad de Ciencias Campus Fuentenueva,Granada,GR Spain18071. E-mail:rjimenez@ugr.esReceived:21May2013.First decision:31May2013.Accepted:5August2013.Ó2013by the Society for the Study of Reproduction,Inc.This is an Open Access article,freely available through Biology of Reproduction’s Authors’Choice option.eISSN:1529-7268ISSN:0006-3363to that of Wnt4null mutants[18].Available data suggest that Rspo1may cooperate with Wnt4to stabilize b-catenin,helping to counteract the establishment of the Sox9/Fgf9positive feedback loop during mouse ovarian development(see reference[19]for a review).Indeed,mice carrying a constitutive active form of b-catenin show XY female sex reversal[20].Before sex determination,Sox9expression occurs at a basal level in both male and female gonadal primordia,but at11.5 days postcoitum(dpc),it is up-regulated in males and down-regulated in females[21].The regulatory action of SRY on Sox9has recently been unraveled.SRY,in a synergistic action with NR5A1/SF1,up-regulates Sox9in male undifferentiated gonads by binding to a testis-specific Sox9enhancer[22]. However,it is not known how Sox9is down-regulated in the female gonad.Although Wnt4null mutant XX gonads can show transient upregulation of Sox9expression[13],null mutations in either the gene for b-catenin(Ctnnb1)or Foxl2are not sufficient to maintain de-repression of Sox9expression in the early XX gonad and neither are compound null mutations involving Wnt4and Foxl2,or Rspo1and Foxl2[23,24](see also our unpublished data).All this suggests that additional genetic elements other than those currently known are involved in sex determination,especially those required to drive ovary and repress testis differentiation.In this context,several studies have suggested that micro-RNAs(miRNAs)are involved in this process,but this hypothesis has not yet been sufficiently explored.MicroRNAs are small,noncoding RNAs that regulate the expression of target genes,either by guiding the cleavage of their target mRNAs or by inhibiting their translation[25–28]. miRNAs have been shown to exert post-transcriptional control of genes involved in development and other biological aspects including disease,cell death,cell proliferation and hematopoi-esis,in a variety of organisms,including Caenorhabditis elegans,flies,mammals and plants[26,29–34].Although no miRNA is currently known to play a role in vertebrate sex determination,there is indirect evidence to suggest that miRNA could be involved in the process:several miRNAs show a sexually dimorphic expression pattern in the mouse gonad at 13.5dpc[35];and targeted disruption of Dicer1,a gene involved in miRNA maturation,has revealed that Dicer protein is necessary to maintain Sertoli cell function[36–38].MATERIALS AND METHODSAnimals and Gonadal CellsGonads were dissected from Swiss,Parkes,and CD1outbred mouse embryos at11.5and13.5dpc and were either fixed or prepared for cell culture. Individual embryos were sexed either by gonad morphology(at13.5dpc)or at earlier indifferent stages using sex chromatin staining of amniotic cells[39]and duplex PCR for zinc finger protein1,Y linked(Zfy1)and the gene for myogenin(Myog).For purification of the RNA used in microarray experiments, a total of24male11.5dpc,19male13.5dpc,31female11.5dpc,and18 female13.5dpc gonads were unambiguously identified,pooled,and processed (see later discussion).For transfection studies,sexed male and female gonads were pooled,disaggregated both mechanically and enzymatically(125l g/ml collagenase;Roche)and cultured either in LabTek chamber slides or in24-well plates,using Dulbecco modified Eagle medium(DMEM;Sigma)with10% fetal calf serum(FCS,Sigma).Similarly,primary chondrocyte cultures were from mouse embryonic limbs.For in situ hybridization,sexed gonads were fixed overnight in4%paraformaldehyde and further immersed in30%sucrose (w/v),embedded in Tissue-Tek optimal cutting temperature compound (Sakura)and sectioned in a cryostat.Mouse housing and handling,as well as laboratory protocols,were approved by the University of Granada Ethics Committee for Animal Experimentation or were carried out under a U.K.Home Office Project License.MicroRNA Profiling of Mouse GonadsTotal RNA from embryonic gonads at11.5and13.5dpc was purified using a miRNeasy mini kit(Qiagen)according to the manufacturer’s protocol. Further steps were performed by Exiqon miRNA Profiling Service.The quality and integrity of the RNA samples was assessed in a Bioanalyser2100(Agilent Technologies),and the concentration was measured with a spectrophotometer (Nanodrop).All samples used for array hybridization showed RNA integrity numbers higher than9.Micro-RNA samples were labeled using a miRCURY Hy3/Hy5power labeling kit from Exiquon.Four embryo gonad samples were analyzed:male11.5dpc,female11.5dpc,male13.5dpc,and female13.5dpc. Each sample was labeled with Hy3and double hybridized against a pool of the four samples labeled with Hy5,which was used as a common reference. Hybridizations were performed with an HS400/4800hybridization station (Exiqon-Tecan).The miRCURY locked nucleic acid(LNA)microarray slides were scanned using a G2565BA microarray scanner system(resolution10l m; Agilent Technologies).Array Slide Quality Control Using Spike-InsHy3and Hy5labeling reactions,hybridization,and performance of array experiment were evaluated with RNA controls(spike-in)added at various concentrations,covering the full signal range,to the labeling reactions.A high correlation for both the Hy3and the Hy5channels indicated that both labeling and hybridization were successful.Each spike-in control had32replicates of capture probes on the array.MicroRNA Microarray Data ValidationData from selected micro-RNAs were validated in our laboratory by real-time RT-PCR using the miRCURY LNA micro-RNA PCR system(Exiqon),in a Chromo4real-time thermocycler(Bio-Rad)according to Exiqon protocols. As primer sets specific for the amplification of miR-124were not available from Exiqon,microarray data from this micro-RNA were validated using TaqMan micro-RNA assays from Applied Biosystems,according to the manufacturer’s procedures.For miR-124,three gene expression quantification experiments were performed with different gonadal RNA samples,and each quantitative(Q)-RT-PCR assay was done in triplicate using the U6snRNA as a reference.MIAME-compliant data were submitted to ArrayExpress accession: E-MEXP-2252,experiment name:Expression of miRNAs in mouse embryo gonad during the critical period of sex determination;specified release date: May11,2013.Identification of Active miR-124GenesRT-PCR was used to identify which of the three possible miR-124 precursor transcripts were expressed in the developing gonads.As the predicted amplification products are short sequences,short oligonucleotides(oligos)were designed with the eprimer3application from the emboss package,and the alignment temperature of the oligos was increased and adjusted,adding unspecific nucleotides at the50end of each oligo.The primers sequences were as follow:forward50-GCC TCT CTC TCC GTG T-30and reverse50-CCA TTC TTG GCA TTC A-30for mmu-miR-124-1;forward50-AGA GAC TCT GCT CTC CGT GT-30and reverse50-CTC CGC TCT TGG CAT TC-30for mmu-mir-124-2;and forward50-GGC TGC GTG TTC ACA G-30and reverse 50-ATC CCG CGT GCC TTA-30for mmu-mir-124-3.One triplicate quantitative(Q)-RT-PCR reaction was carried out using RNA samples from six pooled13.5dpc mouse embryonic gonads of each sex. BioinformaticsMicroarray image analysis was carried out using ImaGene version7.0 software(BioDiscovery,Inc.).The quantified signals(background correction Normexp,with offset value10[40]were normalized by Exiquon miRNA Profiling Service,using the global LOcally WEighted Scatterplot Smoothing (LOWESS)regression algorithm.In order to assess the biological significance of differentially expressed miRNAs,we searched for putative targets in genes involved in sex determination or gonad development,fetching miRNA data from miRbase(/),gene descriptions from Ensembl (/),and filtering the results with Gene Ontology(GO) terms(/)by using Perl scripts(http://www.perl. org/)developed for this purpose.Graphs were drawn and statistics were analyzed with gnu-R software(/).REAL ET AL.Luciferase AssaysHEK293T cells cultured in24-well plates were cotransfected using Lipofectamine2000(Invitrogen),with a miR-124overexpression plasmid vector(1l g/well;ref.MmiR3282-MR04;Genecopoeia),and pLuc-Sox930-untranslated region(UTR)(0.2l g/well).Forty-eight hours after transfection, cells were harvested for luciferase assay.Dual reporter assays were performed by measuring both firefly and Renilla luciferase activities(LucPair miR dual luciferase assay kit;ref.LPFR-M010;Genecopoeia)according to the manufacturer’s protocols.MicroRNA In Situ HybridizationIn situ hybridization experiments with a miR-124-specific miRCURY LNA micro-RNA detection probe(catalog no.33007-01;sequence:CTTGGCATT CACCGCGTGCCTTA;provided at25l M;Exiqon)were performed.Briefly, 8-l m-long cryosections(four per slide)were hybridized with30nM50-digoxigenin-labeled LNA probe overnight at588C.Signals were revealed using anti-digoxigenin alkaline phosphatase-conjugated antibodies(1:1500 dilution;Roche)and bone morphogenic protein(BMP)purple substrate.In situ hybridization experiments were performed six times with different gonad samples.For negative controls,gonad cryosections were subjected to the entire protocol but the probe was omitted from the hybridization mixture. MicroRNA Knock-Down and Overexpression in Cultured CellsPrimary gonadal cell cultures were transfected for miRNA inhibition and overexpression experiments.The inhibition of miR-124in XX embryonic gonad cells was produced with a high-affinity LNA-enhanced probe (antagomir)for specific in vitro knock-down(ref.139452-04;miRCURY LNA micro-RNA inhibitor50-fluorescein-labeled;from Exiqon).As a negative control,we transfected the same type of cells with a different antagomir, specific for miR-144,an miRNA that is not expressed in the female gonad according to our miRNA microarray data.Cells were plated to a density of 50000cells per cm2in8-well chamber slides(LabTek)for immunofluores-cence studies or in24-well culture plates for further RNA extraction,in500l l of DMEM supplemented with10%FCS.For transfection with the silencing probes,cells were transfected for4–6h after plating with a mixture of1.5l l of Lipofectamine2000(Invitrogen)in50l l of Opti-Mem-I reduced serum medium without antibiotics and6l l of probe in50l l of Opti-Mem,according to the guidelines of the manufacturer.After24h,the medium was replaced with DMEM supplemented with10%FCS and15l g/ml tetracycline.Forty-eight hours later the efficiency of transfection was checked by the fluorescence of the probe.The efficiency of transfection was higher than90%in all these experiments.Seventy-two hours after transfection,the cells were fixed in4% formaldehyde or processed for total RNA extraction.Overexpression of miR-124was performed using a precursor miRNA expression clone for the mouse mmu-mir-124-2gene(ref.MmiR3282-MR04; Genecopoeia).In this case,cells transfected with a precursor miRNA-scrambled control clone(ref.CmiR0001-MR04;Genecopoeia)were used as the negative control.For transfection of primary cultures of embryonic gonad cells with these vectors,we followed a procedure similar to that described previously with some exceptions.Before culture,cells were enzymatically disaggregated with collagenase(250l g/ml)and then transfected with1l g of vector by using an electroporator(Nucleofector II;Amaxa),obtaining a transfection efficiency higher than60%,which was determined by counting the percentage of fluorescent cells(green fluorescent protein[GFP]was included in the transfection vector).Forty-eight hours after transfections,cells were either fixed in4%formaldehyde or processed for total RNA extraction.Both knock-down and overexpression experiments were performed more than five times using3–413.5dpc pregnant females per experiment,providing approximately60embryonic gonads,which were divided into three sets of20 gonads each.Cells from the first set were treated with the miR-124-antagomir/ overexpression vector,those from the second set were treated with the miR-144-antagomir/control vector,and those from the third set remained untreated before fixation or RNA extraction.Gene Expression QuantificationTo check the genetic effects of the miRNA knock-down experiments by Q-RT-PCR,total RNA was extracted with Trizol reagent(Qiagen)after transfection and300–900ng total RNA was reverse transcribed using Superscript II(Invitrogen)and random hexamers as primers.RNA samples were purified from both nontransfected and transfected cells with the miR-124-silencing LNA probe.Expression levels of mmu-miR-124were quantified using TaqMan micro-RNA assays(Applied Biosystems)according to the manufacturer’s procedures.Quantification of Sox9expression levels was performed using1-l l aliquots of a1:10dilution of the RT reaction as templates in the Q-PCR reactions.Each PCR reaction was run in triplicate,and all experiments were repeated at least three times.All samples were normalized relative to glyceraldehyde-3-phosphate dehydrogenase(Gapdh),using the 2–DD CT(Livak)method.The primers for Sox9mRNA amplification were forward50-CGG AGG AAG TCG GTG AAG A-30and reverse50-GTC GGT TTT GGG AGT GGT G-30.For Gapdh amplification the primers were: forward50-GGC ATT GCT CTC AAT GAC AA-30and reverse50-TGT GAGG GAG ATG CTC AGT G-30.The identity of the amplified fragments was confirmed by sequencing in both cases.SOX9Protein DetectionImmunofluorescence analyses were performed to check the presence of SOX9protein in transfected gonadal cells.Cells were cultured in LabTek chamber slides,fixed for5min in4%formaldehyde,washed in PBS,blocked for1h at room temperature with2%BSA in PBS,and incubated overnight with a1:100dilution of SOX9-specific rabbit primary antibody(two different antibodies were used,antibody code sc-20095[Santa Cruz Biotechnology]and code AF3075[R&D Systems],and the same results were obtained).Slides were then washed in PBS and incubated for1h at room temperature with a1:500 dilution of Alexa Fluor555goat anti-rabbit secondary antibody(Invitrogen). Sections were then washed in PBS,incubated in a solution of100ng/ml40,6-diamidino-2-phenylindole(DAPI)for30min at room temperature,washed again in PBS,and mounted(Vectashield mounting medium;Vector Laboratories).Images were obtained with an Olympus BX41microscope with epifluorescence equipment.RESULTSMicroRNA Expression Profiling of Mouse11.5-and13.5-dpc Male and Female GonadsAll data from miRNA microarray profiling experiments in the present study are provided in Supplemental Data(Project-Summary.xls;all Supplemental Data are available online at ).Among757miRNAs included in the miRCURY LNA microarray slides,71miRNAs showed differential expression either between sexes or between developmental stages.The expression profiles of these miRNAs are shown in a heat map diagram(Fig.1),as previously described[41],where rows represent miRNAs and columns represent different samples.This diagram also includes a two-way hierarchical clustering of genes and samples.The miRNA clustering tree,constructed based on log2(Hy3:Hy5)ratios(.0.5SD among the four samples), permits identification of five different gene clusters(Fig.1,A–E).The miRNAs in clusters A and E are either down-regulated (Fig.1A)or up-regulated(Fig.1E)during the transition between the two developmental stages(11.5and13.5dpc)but show no great differences in levels of expression between testes and ovaries at13.5dpc.Therefore,these miRNAs are probably not involved in gonadal sex differentiation and thus have little relevance to the current study.In contrast,miRNAs in clusters B,C,and D,show differential expression in a sex-specific fashion at13.5dpc but not at11.5dpc and are therefore good candidates to be involved in gonadal sex differentiation.Cluster B includes3miRNAs that are up-regulated in ovaries and down-regulated in testes at13.5dpc, suggesting a potential role in ovarian development.Cluster C contains six miRNAs that are exclusively up-regulated in13.5 dpc testes,suggesting a role in testis development.On the other hand,cluster D consists of11miRNAs up-regulated only in 13.5dpc ovaries and,therefore,similar to those in cluster B; these miRNAs could be involved in ovarian development.The outcome of our miRNA microarray profiling experi-ments was validated by Q-RT-PCR.The expression levels of at least2representative miRNAs of each cluster,except formiR-124REGULATION OF SOX9IN OVARIAN CELLScluster E,were measured and compared with the corresponding array expression profiles (Fig.2A and Supplemental Fig.S1).In general,relative expression levels in microarrays correspond to those in the Q-RT-PCR assays.Although mmu-miR-126-5p and mmu-miR-103were classified as members of cluster A by the clustering analysis,because differences in expression levels between developmental stages are higher than those between sexes at 13.5dpc,the differential expression in 13.5dpc male and female gonads should be taken into account.Thus,we included these miRNAs in the Q-RT-PCR validation set.Our results confirmed that the expression of both miRNAs is significantly higher in females than in males at 13.5dpc (miR-103also at 11.5dpc),suggesting a potential role in ovarian development.Identification of mmu-mir-124as a Candidate Gene for Ovarian DevelopmentWe performed bioinformatic analyses to investigate the potential biological role of miRNAs that showed sex-specific expression and thus to identify new genetic elements involved in mammalian sex determination.We searched mainly specific miRNA target sequences in the 30-UTR regions of genes associated with GO terms such as,sex determination ,sex differentiation ,gonad differentiation ,testis development ,ovary development ,gonad development ,Sertoli cell differentiation ,and follicle cell differentiation .Using these criteria,we identified miR-124as a good candidate gene for ovarian development because several genes involved in sex determi-nation,including Sox9,share potential targets of this miRNA in their 30-UTR regions.Furthermore,miR-124was included in cluster B (differentially expressed in developing ovaries)and is known to control Sox9expression in the brain [42].These data strongly suggested that miR-124may play an important role in early steps of ovarian development,and thus,we carried out further analyses of this miRNA.The functional interaction between miR-124and Sox9was examined in the luciferase reporter assays.We found that miR-124significantly reduced the activity of the luciferase gene fused to the Sox930UTR by more than 70%,supporting the notion that miR-124can modulate Sox9expression by binding to its 30-UTR (Supplemental Fig.S2).Cheng et al.[42]previously described a similar effect by using a different reporter vector.miR-124is Up-Regulated in the Mouse Female Gonad Between 11.5and 13.5dpcIn order to confirm the expression data reported by our microarray screening for miR-124,we also performed Q-RT-PCR analyses and in situ hybridization with mouse embryonic gonads.Expression quantification by Q-RT-PCR confirmed that miR-124is up-regulated in the ovary but not in the testis during the critical period of gonad differentiation between 11.5and 13.5dpc (Fig.2A).According to miRBase,there are three predicted precursor hairpin sequences of mmu-miR-124in the mouse genome,giving rise to the same mature sequence.The identity of the miR-124precursor(s)expressed in mouse embryonic gonads was investigated by Q-RT-PCR with sets of primers specific for the three miR-124pre-miRNAs.Our results revealed that all three miR-124genes are expressed in 13.5dpc mouse ovaries (mainly pre-miR-124-2)but not in testes of the same developmental stage,where pre-miR-124-1is absent and the other two precursors show low expression levels (Fig.2B).Expression values were always significantly higher in females than in males for the three precursors (P¼FIG. 1.Heat map diagram showing miRNA expression profiles in embryonic gonads during the period of sex determination.A two-way hierarchical clustering of genes and samples identified five different gene clusters.Clusters A and E include miRNAs showing no sex-specific down-regulation (A)or up-regulation (E)during the transition between 11.5and 13.5dpc developmental stages;these genes are thus probably not involved in sex determination.Cluster B contains three miRNAs up-regulated in 13.5-dpc ovaries and down-regulated in 13.5testes,with a potential role in ovarian development.Cluster C includes 6miRNAs up-regulated in 13.5dpc testes but not in 13.5dpc ovaries,with a possible role in testis development.Cluster D contains 11miRNAs up-regulated in 13.5dpc ovaries but not in 13.5dpc testes,suggesting a role in ovarian development.REAL ET AL.FIG.2.Spatiotemporal expression pattern of miR-124in mouse embryonic gonads during the period of sex determination.A )Q-RT -PCR (gray bars)and miRNA microarray (white bars)data of miR-124expression levels in male (M)and female (F)gonads at 11.5and 13.5dpc.miR-124is sex-specifically up-regulated in the 13.5dpc female gonad.B )Q-RT-PCR quantification of the active miR-124precursors 1,2,and 3,in male (gray bars)and female (white bars)embryonic gonads.Amplified products derived from all three precursors are mainly detected in the female samples.C –F )In situ hybridization detection of miR-124in developing mouse gonads.Hybridization signal is very weak in 11.5dpc gonads of both sexes (C and E )and becomes prominent in the coelomic epithelium of the XY gonad and the cortical region of the 13.5dpc XX gonad (D and F ,arrows in photomicrographs).G,gonad;M,mesonephros .Dashed lines mark the border between the gonad and the mesonephros.Bar ¼300l m for all images.miR-124REGULATION OF SOX9IN OVARIAN CELLS0.015for pre-miR-124-1;P¼0.016for pre-miR-124-2;P¼0.010for pre-miR-124-3).In situ hybridization with a miR-124-specific probe showed a faint or very weak expression inboth male and female gonads at11.5dpc(Fig.2,C and E).At 13.5dpc,miR-124expression was prominently up-regulated inthe cortical region of the female gonad,whereas it wassustained,albeit weak,in the coelomic epithelium of the male gonad(Fig.2,D and F).Knock-Down of miR-124Induces Up-Regulation of Sox9in 13.5-dpc Embryonic XX Gonadal CellsBecause the30UTR region of Sox9contains a miR-124 target sequence,we hypothesize that miR-124could be responsible for maintaining down-regulation of Sox9in the female gonad from the sex differentiation stage onward.To test this hypothesis,we transfected13.5dpc embryonic gonadal cells with a specific miR-124-silencing probe,designated antagomir-124or Ant-124,that antagonizes the action of miR-124.Q-RT-PCR analyses demonstrated a significant reduction of endogenous miR-124in cells transfected with the antagomir (Fig.3A).Immunofluorescence analysis revealed that the SOX9protein was absent in the nuclei of XX embryonic gonadal cells when cells were untreated(Fig.3B)or transfected with an unrelated antagomir,Ant-144(Fig.3C).In contrast, SOX9expression was observed in XX gonadal cells transfected with Ant-124(Fig.3D),such that they look similar to control XY gonadal cells(Fig.3E).Q-RT-PCR showed a significant increase in Sox9mRNA in XX gonadal cells transfected with Ant-124but neither in untreated XX cells nor in those transfected with Ant-144(Fig.3F).Overexpression of miR-124Induces Down-Regulation of Sox9in Embryonic Chondrocytes but not in XY Gonadal CellsBased on our hypothesis,overexpression of miR-124would be expected to induce down-regulation of Sox9in cells expressing this gene naturally.To test this possibility,we transfected both cultured chondrocytes and XY gonadal cells with either a precursor miRNA expression vector containing the mouse mmu-mir-124-2gene or a negative control vector containing a scrambled miRNA sequence.The levels of miR-124increased considerably in cells transfected with mmu-mir-124-2,as measured by Q-RT-PCR(Fig.4A).Immunofluores-cence analyses demonstrated that the percentage of SOX9-positive cells was significantly lower in chondrocytes trans-fected with the mmu-mir-124-2expression vector than in those with the control vector.However,this effect was not observed in XY gonadal cells;the percentage of cells expressing SOX9 did not decrease in the presence of the mmu-mir-124-2 expression vector(Fig.4,B–F).Consistent with the results of immunofluorescence studies,Q-RT-PCR analyses showed that overexpression of mmu-mir-124-2reduces the expression levels of Sox9mRNA in chondrocytes,whereas no significant differences were observed in XY gonadal cells(Fig.4G). Because SOX9expression is already robust and maintained by several feedback loops in13.5dpc mouse embryonic testes, miR-124might not be able to efficiently overcome SOX9 expression.Therefore,we repeated the same set of experiments using11.5dpc XY gonadal cells,where Sox9transcription has just started.However,we obtained similar results,confirming the fact that overexpression of miR-124is not sufficient to affect SOX9expression in XY gonadal cells(Supplemental Fig.S3).DISCUSSIONDespite the discovery of the mammalian testis-determining gene SRY some20years ago[1,2],it is thought that some regulatory elements remain unknown,particularly those involved in determining the ovary.Because miRNAs are involved in many developmental processes,it is reasonable to suspect that they may also have some role in gonad development.Several recent studies strongly suggest that this is the case in mammals[35–38].Using miRNA microarray technology,we identified22miRNAs(including mmu-miR-126-5p,mmu-miR-103,and clusters B,C,and D)that are either up-or down-regulated during the critical period of sexual differentiation in a sex-specific manner,indicating that they could participate in gonadal sex differentiation(Fig.1). Interestingly,coincident with ovarian cell determination at 13.5dpc,most of them(16)are up-regulated in the ovary at this stage,suggesting a potential role in ovarian development.Using a clone-based miRNA amplification profiling meth-od,Takada et al.[35]reported that some miRNAs show a sex-dependent expression pattern in13.5dpc mouse gonads. Concerning some of the miRNAs identified by those authors, their results contradict those of our microarray-based screening. Notably,Takada et al.reported that the expression of miR-124 is higher in developing testes than in ovaries[35].However, our data,particularly for miR-124expression,have been repeatedly validated by Q-RT-PCR and in situ hybridization, which is not included in studies of Takada et al.In addition,a recent publication reported that miR-202-5p/3p are differen-tially expressed in the developing testis[43],thus coinciding with our microarray data.In fact,miR-202-3p is included in cluster C and miR-202-5p is in cluster E because the higher expression detected in the testis did not exceed the SD.0.5 threshold(Fig.1).This represents further validation of our microarray results.Several findings support the fact that miR-124could have an important role during ovarian development.First,our miRNA microarray profiling showed that miR-124is differ-entially up-regulated in the XX but not the XY gonad during the transition from11.5to13.5dpc stages.Second,the computational analysis for the miRNAs selected from our microarray results identified potential targets for miR-124in the30-UTR regions of four genes of the male pathway in both human and mouse:Sox8(the SRY-box containing gene8), Sox9,Dmrt1,and AR(androgen receptor).Third,Cheng et al.[42]showed that the mouse miR-124regulates adult neurogenesis by repressing SOX9production in the subven-tricular zone of the brain[42],raising the possibility that this miRNA could act also in the gonad.Overall,these findings strongly suggested that the main role of miR-124in the ovary would be to inhibit the production of SOX9protein.To verify this hypothesis,miR-124was knocked down or overexpressed in primary cultures of embryonic gonadal cells. Transfection with an miR-124-specific antagomir induced the ectopic production of SOX9protein in XX gonadal cells,as well as a significant increase in Sox9mRNA/transcript, implying that miR-124actively represses Sox9in female gonadal cells.The increased expression levels of Sox9in XX cells transfected with antagomir-124are lower than those observed in XY gonadal cells,suggesting that SRY-mediated up-regulation of Sox9is required to reach the male expression level.In the developing mouse testis,Sox9is activated primarily,probably by SF1,in both XY and XX gonads prior to sex differentiation[44].At the time of sex determination, Sox9is up-regulated in the XY gonad by the combined, synergistic action of SRY and SF1on a testis-specific Sox9REAL ET AL.。

FGF21对非酒精性脂肪肝影响的研究进展

FGF21对非酒精性脂肪肝影响的研究进展发布时间：2022-11-14T07:17:12.912Z 来源：《医师在线》2022年6月12期作者：王浩田桦卓越通讯作者[导读]FGF21对非酒精性脂肪肝影响的研究进展王浩田桦卓越通讯作者（佳木斯大学附属第一医院；黑龙江佳木斯154000）摘要：非酒精性脂肪性肝病（NAFLD）现在被认为是全球最常见的肝病。

它涵盖了广泛的疾病，从简单的脂肪变性，到非酒精性脂肪性肝炎，再到纤维化，最终是肝硬化和肝细胞癌。

目前，NAFLD已被证实与血脂异常、胰岛素抵抗和糖尿病前期相关，而这些通常被归为代谢综合征，对普通人群的未来健康构成严重风险[1]。

NAFLD的标志是肝细胞中甘油三酯(TG)过高，这不是由过量的酒精引起的。

肝细胞内的慢性血脂异常可进一步导致肝脏损伤，导致NAFLD并发症，涉及心脏、肾脏等多个器官。

此外，NAFLD包含多种相互作用因素，其中胰岛素抵抗、内脏脂肪、肝脏血脂异常、内皮功能障碍等均可能参与NAFLD的发病机制[2]。

在NAFLD状态下，促炎条件及相关代谢综合征不可避免地诱导细胞因子的异常分泌，而其中一种细胞因子是成纤维细胞生长因子21 (FGF21)。

关键词：成纤维细胞生长因子21，非酒精性脂肪性肝病，糖与脂肪代谢。

中图分类号：R574 文献标识码：A 1.FGF家族FGF家族包含大量参与细胞生长、细胞分化和胚胎发育等多种作用的因子。

大多数FGF体积较小，分子量范围为17-34 kDa。

在FGF多肽与质膜上表达的几种酪氨酸激酶受体(FGFRs)之一结合后，信号传导得以传播。

FGFs被分为7个亚家族，尽管一些研究人员将FGF3分为自己独特的亚家族[3]。

激素型FGFs组成了其中一个亚家族，包括三个FGF19(和小鼠同源Fgf15)， FGF21和FGF23。

如上所述，这个亚家族的成员是不同的，因为他们缺乏肝素结合结构域，需要专性的辅助受体来传递信号:FGF19和21的KLB和FGF23的klotho。