A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar
三阴性乳腺癌组织中FOXJ1、SATB1的表达变化及临床意义

比例<50%记2分,50%臆阳性细胞比例<75%记 3分,阳性细胞比例逸75%记4分。根据染色强度将 切片分为4个等级,无色记0分,淡黄色记1分,棕黄 色记2分,棕褐色记3分,最后将阳性细胞数评分和 染色强度评分相乘得到最终评分并按等级分为阴性 和阳性,其中0~4分为阴性、5~12分为阳性㈣。
老年痴呆症小鼠模型构建原理

老年痴呆症小鼠模型构建原理
1974年,Rudolf Jaenisch通过将SV40 病毒的DNA注射到小鼠的囊胚中,创造了第一只携带外源基因的小鼠。
后来又有研究人员把Murine leukemia病毒注射到小鼠胚胎得到了能通过生殖系统稳定遗传的小鼠,并且外源基因能在后代中稳定表达。
这些能稳定遗传且表达外源基因的小鼠即我们一般意义上所说的转基因小鼠。
下面是,老年痴呆症小鼠模型介绍
阿尔兹海默症(AD)小鼠模型,APP/PS1小鼠,APP/PS1双转基因的老年痴呆症小鼠模型。
可用于老年痴呆症和神经退行性变化的神经生物学研究、老年痴呆症的人/鼠基因同系物研究、老年痴呆症的干预治疗及药物研发。
关于抗衰老研究进展的英文原文文献

Aging by Telomere Loss Can Be ReversedBruno Bernardes de Jesus 1and Maria A.Blasco 1,*1Telomeresand Telomerase Group,Molecular Oncology Program,Spanish National Cancer Centre (CNIO),Melchor Ferna´ndez Almagro 3,Madrid E-28029,Spain*Correspondence:mblasco@cnio.es DOI 10.1016/j.stem.2010.12.013Recently in Nature ,Jaskelioff et al.(2010)demonstrated that multiple aging phenotypes in a mouse model of accelerated telomere loss can be reversed within 4weeks of reactivating telomerase.This raises the major question of whether physiological aging,likely caused by a combination of molecular defects,may also be reversible.Accumulation of short/damaged telo-meres with increasing age is considered one of the main sources of aging-associ-ated DNA damage responsible for the loss of regenerative potential in tissues and during systemic organismal aging (Harley et al.,1990;Flores et al.,2005).Mounting evidence suggests that telome-rase is a longevity gene that functions by counteracting telomere attrition.Thus,telomerase-deficient mice age prema-turely,and telomerase overexpression results in extended longevity in mice (Tomas-Loba et al.,2008).Moreover,human mutations in telomerase compo-nents produce premature adult stem cell dysfunction and decreased longevity (Mitchell et al.,1999).Previous work had shown that restora-tion of telomerase activity in mouse zygotes with critically short telomeres,owing to a deficiency in the telomerase RNA component (Terc),rescues critically short telomeres and chromosomal instability in the resulting mice (Samper et al.,2001).Restoration of telomerase activity in zygotes also pre-vented the wide range of degenerative pathologies that would otherwise appear in telomerase-deficient mice with critically short telomeres,including bone marrow apla-sia,intestinal atrophy,male germ line depletion,and adult stem cell dysfunction (Samper et al.,2001;Siegl-Cachedenier et al.,2007),and resulted in a normal organismal life-span (Siegl-Cachedenier et al.,2007).Together,all the above find-ings indicate that aging provoked by crit-ical telomere shortening can be prevented or delayed by telomerase reactivation.From these grounds,reversion of aging caused by telomere loss was the next frontier.A recent study in Nature takes an important step forward from these previous findings by using a new mouse model for telomerase deficiency,de-signed to permit telomerase reactivation in adult mice after telomere-induced aging phenotypes have been established (Jas-kelioff et al.,2010).Specifically,DePinho and colleagues generated a knockin allele encoding a 4-OH tamoxifen (4-OHT)-inducible mouse telomerase (TERT-ER)under the control of the TERT endogenous promoter.In the absence of tamoxifen,these mice exhibit premature appearance of aging pathologies and reduction in survival (Figure 1).These mice phenocopy previously described Terc -deficient mice,which highlights that elongation of short telomeres by telomerase is the main mechanism by which telomerase protects from aging pathologies.Importantly,4weeks of tamoxifen treatment to induce TERT re-expression in adult TERT-ER mice with clear signs of premature aging was sufficient to extend their telomeres and rescue telomeric DNA damage signaling and associated check-point responses.Dramatically,tamox-ifen-induced TERT re-expression also led to resumption of proliferation in quies-cent cultured cells and eliminated the degenerative phenotypes across multiple organs,including testis,spleen,and intes-tines (Figure 1).Reactivation of telome-rase also ameliorated the decreased survival of TERT-ER mice.These findings represent an important advance in the aging field,as they show that aging induced by telomere loss can be reversed in a broad range of tissues and cell types,including neuronal function.Looking to the future,the next key question is to whatextent natural,physiological aging is caused by the pres-ence of critically short telo-meres and,consequently,to what extent telomere restora-tion will be able to reverse physiological aging.In this re-gard,other recent findings support the idea that telomere shortening does impactnatural mouse aging.On onehand,despite the long-standing belief that mouseaging was not linked to telo-mere shortening giventhat Figure 1.Antiaging Effects of Telomerase Schematic showing the major findings of Jaskelioff et al.(2010).Telomerasereactivation in late generation telomerase-deficient mice (G4TERT-ER )couldrevert some of the aging phenotypes observed,demonstrating the regenera-tive potential capacity of different tissues.Cell Stem Cell 8,January 7,2011ª2011Elsevier Inc.3Cell Stem CellPreviewsmice are born with very long telomeres—much longer than human telomeres—mouse telomeres do suffer extensive shortening associated with aging(Flores et al.,2008).In particular,while mouse cells maintain relatively long telomeres during theirfirst year of life,there is a dramatic loss of telomeric sequences at2years of age,even in various stem cell populations, and this change is concomitant with the loss of regenerative capacity associated with mouse aging.In addition,telome-rase-deficient mice from thefirst genera-tion(G1TercÀ/À)exhibit a significant decrease in median and maximum longevity and a higher incidence of age-related pathologies and stem cell dysfunc-tion compared with wild-type mice(Flores et al.,2005;Garcia-Cao et al.,2006),indi-cating that,as in humans,telomerase activity is rate limiting for natural mouse longevity and aging.These results suggest that strategies aimed to increase telome-rase activity may delay natural mouse aging.Further supporting this notion,it was recently shown that overexpression of TERT in the context of mice engineered to be cancer resistant owe to increase expression of tumor suppressor genes(Sp53/Sp16/SARF/TgTERT mice)wassufficient to decrease telomere damagewith age,delay aging,and increase medianlongevity by40%(Tomas-Loba et al.,2008).However,it remains to be seenwhether telomerase reactivation late in lifewould be sufficient to delay natural mouseaging and extend mouse longevity withoutincreasing cancer incidence.In summary,these proof-of-principlestudies using genetically modified miceare likely to encourage the developmentof targeted therapeutic strategies basedon reactivation of telomerase function.Indeed,small molecule telomerase acti-vators have been reported recently andhave demonstrated some preliminaryhealth-span beneficial effects in humans(Harley et al.,2010).Identifying drugabletargets and candidate activators clearlyopens a new window for the treatment ofage-associated degenerative diseases.REFERENCESFlores,I.,Cayuela,M.L.,and Blasco,M.A.(2005).Science309,1253–1256.Flores,I.,Canela,A.,Vera,E.,Tejera,A.,Cotsare-lis,G.,and Blasco,M.A.(2008).Genes Dev.22,654–667.Garcia-Cao,I.,Garcia-Cao,M.,Tomas-Loba,A.,Martin-Caballero,J.,Flores,J.M.,Klatt,P.,Blasco,M.A.,and Serrano,M.(2006).EMBO Rep.7,546–552.Harley, C.B.,Futcher, A.B.,and Greider, C.W.(1990).Nature345,458–460.Harley, C.B.,Liu,W.,Blasco,M.,Vera, E.,Andrews,W.H.,Briggs,L.A.,and Raffaele,J.M.(2010).Rejuvenation Res.14,in press.Publishedonline September7,2010.10.1089/rej.2010.1085.Jaskelioff,M.,Muller,F.L.,Paik,J.H.,Thomas,E.,Jiang,S.,Adams,A.C.,Sahin,E.,Kost-Alimova,M.,Protopopov,A.,Cadinanos,J.,et al.(2010).Nature.10.1038/nature09603.Mitchell,J.R.,Wood,E.,and Collins,K.(1999).Nature402,551–555.Samper,E.,Flores,J.M.,and Blasco,M.A.(2001).EMBO Rep.2,800–807.Siegl-Cachedenier,I.,Flores,I.,Klatt,P.,andBlasco,M.A.(2007).J.Cell Biol.179,277–290.Tomas-Loba, A.,Flores,I.,Fernandez-Marcos,P.J.,Cayuela,M.L.,Maraver,A.,Tejera,A.,Borras,C.,Matheu,A.,Klatt,P.,Flores,J.M.,et al.(2008).Cell135,609–622.HGPS-Derived iPSCs For The AgesTom Misteli1,*1National Cancer Institute,NIH,Bethesda,MD20892,USA*Correspondence:mistelit@DOI10.1016/j.stem.2010.12.014In this issue of Cell Stem Cell,Zhang et al.(2011)generate patient-derived iPSCs for one of the major prema-ture aging diseases,Hutchinson-Gilford Progeria Syndrome(HGPS).These cells are a much-needed new tool to study HGPS,and their use may lead to novel insights into mechanisms of aging.Some problems in biology are more difficult to study than others.Human aging is certainly one of them.Most conclusions regarding molecular mecha-nism of human aging rely on mere correla-tion,and direct experimental testing is generally not feasible.One approach to dissect the molecular basis of human aging is to study naturally occurring premature aging disorders.One of the most dramatic and prominent of such diseases is Hutchinson-Gilford ProgeriaSyndrome(HGPS).Zhang et al.(2011)now report the generation of inducedpluripotent stem cells(iPSCs)fromHGPS cells,providing a powerful newtool to unravel the molecular and physio-logical mechanisms of premature andnormal aging.HGPS is a truly remarkable disease inmany ways.To start with,it affects anunusually wide spectrum of tissues andleads to the development of highly diversesymptoms ranging from depletion ofsubcutaneous fat to loss of hair andtendon contractures.The diversity ofaffected tissues pointed early on to stemcell defects as a likely disease mecha-nism.Most relevant in patients arevascular defects and recurring strokes,which invariably are fatal in patients intheir mid-to late teens(Hennekam,2006).The disease is exceedingly rare4Cell Stem Cell8,January7,2011ª2011Elsevier Inc.Cell Stem Cell Previews。
3变量的转基因动物模型

3变量的转基因动物模型很多研究证明有好几种基因改造的小鼠模型在研究AD的病理机制上是非常有用的。
但是,没有单单一种动物模型是能够准确的代表AD的全部神经病理特征的。
虽然APP转基因小鼠模型已经能够成功的复制Aβ空斑,而突变PS2等位基因模型也能够展示AD的相似病变过程,但是它们都不能够建立起NFT(Howlett andRichardson 2009)。
相似的情况,尽管tau转基因小鼠模型能够呈现tau在AD病变的作用,但却不能复制Aβ空斑,而Aβ空斑却是AD最基本的病理变化(Tanemura et al. 2002).。
若要同时建立有Aβ空斑和NFT特征的模型,就得将几种改造的基因同时注入同一只小鼠中。
Lewis等(Lewis et al. 2001).就尝试在PS1M1461基因敲除小鼠模型中同时注入来自于突变体的组成物基因(APPswe)和mThy1.2启动子驱动的P301L tau改造基因;模型建造十分成功,不仅比起杂交在实验速度上快很多,而且并不改变小鼠的基因型。
这种3变量的转基因动物模型包含了3种突变基因:表达Aβ前体蛋白的基因,表达早老素1的基因和表达tau蛋白的基因。
这种模型能够充分反应了AD中有关Aβ空斑、NFT和tau引起的病理变化(Lewis et al. 2001).。
例如,表达人类tau蛋白的纯合子转基因小鼠模型能够出现Aβ空斑却不能产生NFT。
然而,注射了Aβ基因的mThy1.2启动子驱动的P301L小鼠模型在Aβ注射18天后能够产生相当于平时5倍之多的NFT(Gotz et al. 2001).。
这进一步显示了Aβ和tau蛋白之前联系。
为了进一步证实Aβ和tau之间的联系,Lewis等(2000)设计了APP Tg2576转基因和mThy1.2启动子驱动的P301L交叉小鼠模型。
这种动物模型与单单tau转基因小鼠模型相比,在嗅球和扁桃体里NFT的量增加了7倍。
然后,tau蛋白在单变量和双变量转基因动物模型中的表达量却没有明显改变(Lewis et al.2001)。
乳腺癌患者接受TEC方案与EC-T方案治疗后白细胞水平的变化

乳腺癌患者接受TEC方案与EC-T方案治疗后白细胞水平的变化秦雪【摘要】目的研究乳腺癌患者接受TEC和EC-T化疗方案治疗后白细胞水平变化.方法选取106例乳腺癌患者作为研究对象,按照随机数字表法分为2组,对照组采用多西紫杉醇(T)、表阿霉素(E)、环磷酰胺(C)联合(TEC方案)化疗治疗6周期,观察组采用序贯(EC-T方案)化疗治疗4周期.对比化疗前后2种方案对患者的白细胞水平改变及白细胞下降率的影响.结果治疗7~14天患者白细胞水平降低最为明显,治疗的第7、14、21天2组白细胞水平均下降,但观察组略高于对照组(P<0.05);治疗的第7、14、21天观察组白细胞减少症发生率分别为18.52%、25.93%、37.03%,低于对照组的28.85%、44.23%、65.38%,差异有统计学意义,P<0.05;取4个治疗周期白细胞水平比较,观察组均略高于对照组,组间比较差异有统计学意义,P<0.05.结论 TEC治疗方案与EC-T方案相比,患者白细胞水平降低更严重,建议在病情允许的情况下选择EC-T方案化疗,以减少化疗引起的白细胞降低并发症,促进病情恢复.%Objective To study the change of leukocyte level in patients with breast cancer after treatment with TEC and EC-T chemotherapy.Methods 106 cases of breast cancer patients,according to the random number table method,were divided into 2 groups,the control group(n=52)was treated with docetaxel(T),epirubicin(E),and cyclophosphamide(C)(TEC)chemotherapy for 6 cycles,the observation group(n=54)received sequential EC-T chemotherapy for 4 cycles,change of white blood cells and white blood cells descent rates of the 2 groups before and after chemotherapy were compared.Results White blood cell in7~14 d of treatment were significantly reduced,white blood cell levels of the 2 groups at 7 d,14 d,21 d were decreased,but the observation group was slightly higher than that of the control group(P<0.05);the incidence of leukopenia at 7 d,14 d,21 d of the observation group were respectively 18.52%and 25.93%and 37.03%,which were lower than the control group,28.85%and 44.23%,65.38%,P<0.05,the difference was significant;white blood cell level of 4 treatment cycles were compared,the observation group were slightly higher than the controlgroup,P<0.05.Conclusion TEC regimen has more serious leukopenia than EC-T scheme,it is recommended to select EC-T chemotherapy if the condition allows,in order to reduce chemotherapy-induced leukopenia and promote recovery.【期刊名称】《实用癌症杂志》【年(卷),期】2017(032)009【总页数】4页(P1464-1466,1493)【关键词】乳腺癌;TEC化疗;EC-T化疗;白细胞【作者】秦雪【作者单位】065000 河北省廊坊市人民医院【正文语种】中文【中图分类】R737.9乳腺癌为女性常见恶性肿瘤,发病率高,治疗以综合治疗法为主,比如乳腺癌根治手术后,以化学疗法或放射治疗来巩固治疗,达到减少复发、防止肿瘤扩散或转移、抑制癌细胞的效果[1]。
文档:肿瘤相关巨噬细胞

肿瘤相关巨噬细胞研究进展免疫细胞浸润是肿瘤恶化的标志之一,在所有免疫细胞中,肿瘤相关巨噬细胞是免疫系统和肿瘤的相互作用过程中最关键的调控中心。
肿瘤相关巨噬细胞是实体肿瘤中大量存在的一种白细胞,在大多数人类肿瘤中,肿瘤相关巨噬细胞的浸润和肿瘤相关巨噬细胞相关基因的上调表达严重影响肿瘤的预后和治疗效果, 。
本文将对肿瘤相关巨噬细胞的促肿瘤功能、肿瘤相关巨噬细胞功能的调控以及针对肿瘤相关巨噬细胞的肿瘤免疫治疗进展加以综述。
1. 肿瘤相关巨噬细胞的促肿瘤功能:有研究表明,巨噬细胞吞噬了凋亡的肿瘤细胞之后,肿瘤DNA被并入巨噬细胞的细胞核中,这些巨噬细胞会受到肿瘤DNA的影响,并转变成具有致瘤能力的类似于肿瘤干细胞特性的细胞,但在这类细胞的表面,仍含有巨噬细胞标记。
研究人员把这类受到肿瘤DNA 影响而获得类似肿瘤干细胞样特性的巨噬细胞,称之为Tumacrophage。
肿瘤相关巨噬细胞可以大量分泌促血管新生因子,如血管内皮生长因子VEGF,后者促进肿瘤的血管生成和血源性细胞转移过程,从而可以通过免疫和非免疫过程促进肿瘤的生长。
此外,肿瘤相关巨噬细胞可以通过抑制抗肿瘤免疫反应达到促肿瘤的效果。
如肿瘤相关巨噬细胞产生免疫抑制因子IL-10、TGFb、PGE2等,其中IL-10通过抑制化疗引起的抗肿瘤免疫,显著抑制了该抗肿瘤治疗的效果。
在前列腺癌中,较高的肿瘤相关巨噬细胞浸润预测总体生存率较差,但无生化复发或无复发生存;相反,巨噬细胞清道夫受体1的水平升高与无复发生存率更高有关。
2. 肿瘤相关巨噬细胞功能的调控:大多数情况下,肿瘤相关巨噬细胞主要来自于血液中的单核细胞,通过CCL2趋化因子被招募到肿瘤部位。
最近的报道证明,肿瘤巨噬细胞的主要来源是CCR2+单核细胞,占据了肿瘤内40%的CD45+细胞。
在到达肿瘤病灶的过程中,单核细胞发生一系列变化到达不同组织的肿瘤后,形成的肿瘤相关巨噬细胞类群在表型和功能上也各不相同,调控这一过程的信号主要来自于肿瘤和组织两部分。
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阿尔茨海默病模型小鼠凋亡相关蛋白的表达

阿尔茨海默病模型小鼠凋亡相关蛋白的表达王晓映1刘鹏朱华徐艳峰马春梅代小伟刘颖秦川(中国医学科学院实验动物研究所北京协和医学院比较医学中心,北京100021)〔摘要〕目的探讨阿尔茨海默病(AD )凋亡相关蛋白的表达情况。
方法取3月龄和6月龄APPswe /PS ΔE9小鼠脑组织,利用Western 印迹的方法检测APPswe /PS ΔE9小鼠和对照小鼠脑组织中bcl-2、caspase-9以及caspase-3的表达情况。
结果与同月龄的对照小鼠相比,APPswe /PS ΔE9转基因小鼠中,bcl-2表达水平明显降低,而caspase-9和caspase-3表达水平明显升高。
结论凋亡相关蛋白的异常表达可能在AD 的发生发展过程中起到一定作用。
〔关键词〕阿尔茨海默病;bcl-2;APPswe /PS ΔE9小鼠〔中图分类号〕R749〔文献标识码〕A〔文章编号〕1005-9202(2012)07-1446-02;doi :10.3969/j.issn.1005-9202.2012.07.0561山东大学第二医院病理科通讯作者:秦川(1959-),女,教授,博士生导师,主要从事神经系统退行性病变研究。
第一作者:王晓映(1981-),女,医师,医学博士,主要从事神经系统退行性病变研究。
阿尔茨海默病(AD )是一种神经系统退行性疾病,是导致老年人痴呆的主要原因,其特征性的病理改变是在大脑皮质和海马区域出现老年斑和神经原纤维缠结。
目前,AD 的发病机制仍然不明确,一些研究发现,凋亡的异常可能在AD 的发生发展过程中起到重要作用。
本实验以APPswe /PS ΔE9模型小鼠作为研究对象,探讨AD 中凋亡相关蛋白的表达情况。
1材料与方法1.1APPswe /PS ΔE9转基因小鼠的建立APPswe /PS ΔE9转基因小鼠由中国医学科学院实验动物研究所构建并提供〔1〕。
APPswe 转基因编码一段人鼠嵌合的蛋白,其中APP 的胞外和胞内区域为鼠源性序列,而A β结构域为人源性序列并包含瑞士突变K594N /M595L 。
非洲猪瘟病毒A137R蛋白多克隆抗体的制备与应用

中国预防兽医学报Chinese Journal of Preventive Veterinary Medicine第43卷第1期2021年1月V ol.43No.1Jan.2021doi :10.3969/j.issn.1008-0589.202003013非洲猪瘟病毒A137R 蛋白多克隆抗体的制备与应用向志达1,2,张涛清2,张元峰2,黄丽2,杨玉莹1*,翁长江1,2*(1.长江大学动物科学学院,湖北荆州434025;2.中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/基础免疫创新团队,黑龙江哈尔滨150069)摘要:非洲猪瘟(ASF )是由非洲猪瘟病毒(ASFV )感染引起的一种高致病性病毒性传染病。
为制备兔抗ASFVA137R 蛋白多克隆抗体,本研究以ASFV Pig/HLJ/2018分离株基因组DNA 为模板扩增ASFV A137R 基因,将其克隆至原核表达载体pET-21a (+)中,构建重组表达质粒pET-21a-A137R ,转化大肠杆菌BL21(DE3)感受态细胞后经IPTG 诱导表达,SDS-PAGE 检测结果显示,表达的A137R 重组蛋白分子质量约15.0ku ,其主要以可溶形式表达。
利用HisTrap 亲和层析和凝胶过滤层析纯化获得高纯度的A137R 重组蛋白,利用该纯化重组蛋白免疫新西兰大白兔,制备抗ASFV A137R 蛋白多克隆抗体,经Protein G 亲和层析介质纯化后经western blot 、间接免疫荧光和免疫共沉淀试验检测结果显示,制备的兔抗ASFV A137R 蛋白的多克隆抗体能特异性地识别HEK293T 细胞中瞬时表达的ASFV Flag-A137R 蛋白和ASFV 感染猪肺泡巨噬细胞中表达的ASFV A137R 蛋白。
本研究为进一步探究ASFV A137R 蛋白的功能、其在ASFV 感染及致病性中的作用和诊断技术研究奠定了基础。
关键词:非洲猪瘟病毒;A137R 蛋白;多克隆抗体中图分类号:S852.4文献标识码:A文章编号:1008-0589(2021)01-0060-05Preparation and application of polyclonal antibody against Africanswine fever virus-encoded A137R proteinXIANG Zhi-da 1,2,ZHANG Tao-qing 2,ZHANG Yuan-feng 2,HUANG Li 2,YANG Yu-ying 1*,WENG Chang-jiang 1,2*(1.College of Animal Science,Yangtze University,Jingzhou 434025,China;2.Harbin Veterinary Research Institute,Chinese Academy ofAgricultural Sciences,State Key Laboratory of Veterinary Biotechnology,Harbin 150069,China)Abstract :African swine fever (ASF)is a highly pathogenic viral infectious disease caused by African swine fever virus.To prepare rabbit polyclonal antibody against ASFV A137R protein,the genomic DNA isolated from ASFV Pig/HLJ/2018strain was used as a template to amplify ASFV A137R gene.The target gene was then subcloned into the bacterial expression vector pET-21a to construct the recombinant plasmid pET-21a-A137R.The recombinant plasmid was transformed into E.coli BL21(DE3)and induced by the addition of IPTG to express A137R protein.SDS-PAGE result showed that the recombinant protein was expressed in a soluble form with a molecular mass of 15.0ku.The recombinant protein A137R was purified by HisTrap affinity chromatography and gel filtration chromatography.Two New Zeal white rabbits were immunized with the purified recombinant protein A137R to prepare anti-ASFV A137R polyclonal antibody,and the antibody was then purified by a protein G affinity chromatography column.The results of western blot,indirect immunofluorescence assay and immunoprecipitation showed that收稿日期:2020-03-10基金项目:非洲猪瘟病毒感染与致病机制(31941002、CAAS-ZDXT2018007)作者简介:向志达(1994-),男,湖北宜昌人,硕士研究生,主要从事非洲猪瘟病毒蛋白功能研究.*通信作者:E-mail :**********************;*********************.cn*Corresponding author向志达,等.非洲猪瘟病毒A137R蛋白多克隆抗体的制备与应用第1期非洲猪瘟(African swine fever,ASF)是由ASF病毒(ASFV)感染引起的一种高致病性病毒性传染病,ASFV急性感染可导致家猪死亡率接近100%[1]。
Nestin、ER与Ki67在儿童畸胎瘤组织中的表达及其意义

南昌大学医学院硕士学位论文Nestin、ER与Ki-67在儿童畸胎瘤组织中的表达及其意义姓名:***申请学位级别:硕士专业:儿外科指导教师:***20070601Nestin、ER与Ki-67在儿童畸胎瘤组织中的表达及其意义作者:戴康临学位授予单位:南昌大学医学院1.期刊论文薛黎萍.丁鹏.吴开力.江春光.胡竹林.肖丽波.刘海.Eng-Ang Ling.Li-Ping Xue.Peng Ding.Kai-Li Wu.Chun-Guang Jiang.Zhu-Lin Hu.Li-Bo Xiao.Hai Liu.Eng-Ang Ling Nestin在视神经横断大鼠视网膜Müer细胞上的诱导表达-国际眼科杂志2009,09(3)目的:探讨中间丝蛋白nestin在视神经横断大鼠视网膜神经胶质细胞中的表达情况及可能的意义.方法:制作大鼠视神经横断模型,分别于术后1,3,7d取材,制作视网膜矢状位冰冻切片,进行GS/nestin,免疫荧光双标.提取视网膜总RNA行nestin Real-time PCR半定量分析.结果:正常大鼠视网膜中几乎看不到nestin阳性染色.术后1d,在Müer细胞上出现nestin的诱导表达,术后3d,nestin在Müler细胞上的表达进一步增强,术后1wk,nestin在Müer细胞上的表达保持在较高水平,Real-time PCR半定量分析与以上结果吻合.结论:视神经横断后nestin在Müer细胞上的诱导表达是Müer细胞对视网膜损伤产生的一种反应,Nestin的表达量在一定时间内与病程进展相一致.Nestin在Müer细胞上的诱导表达尤其在足板处的强烈表达可能对视网膜神经节细胞具有保护作用.2.期刊论文黄金狮.戴康临.邹音.杨文萍.陶强.张淑正.肖强.钟华生.邓庆强.陈快.HUANG Jin-shi.DAI Kang-lin. ZOU Yin.YANG Wen-ping.TAO Qiang.ZHANG Shu-zheng.XIAO Qiang.ZHONG Hua-sheng.DENG Qing-qiang.CHEN KuaiER、Ki-67与Nestin在儿童未成熟畸胎瘤组织中的表达及临床意义-江西医学院学报2009,49(2)目的 研究雌激素受体(estrogen receptor,ER)、Ki-67与巢蛋白(Nestin)在儿童未成熟畸胎瘤组织中的表达及其临床意义.方法 制作35例不同级别儿童畸胎瘤手术病理标本组织芯片,采用免疫组化二步法测定畸胎瘤组织中ER、Ki-67与Nestin的表达水平.患儿男21例,女14例.病理分级未成熟畸胎瘤1级11例,2级13例,3级11例.选取成熟畸胎瘤15例作为对照.所有患儿手术前、后均未进行化疗和放疗.随访内容为肿瘤有无复发、转移,有无大小便失禁、尿潴留等.结果 ER、Ki-67、Nestin在不同病理分级畸胎瘤神经上皮组织中随分级升高表达增强,比较差别有统计学意义,在各病理分级儿童畸胎瘤内、中、外胚层(除了神经上皮)组织中表达差异均无统计学意义.随访到29例,2例复发,其余健在,无复发、转移.结论 在儿童未成熟畸胎瘤神经上皮组织中,ER、Ki-67、Nestin的表达与病理分级有关,可作为预后判断的参考指标.未成熟畸胎瘤术后化疗指征有待进一步探讨.3.期刊论文周立兵.李东培.徐杰.阮奕文.姚志彬大鼠基底前脑Nestin阳性神经元表达雌激素α受体-郧阳医学院学报2005,24(4)目的:探讨雌激素对基底前脑巢蛋白(Nestin)阳性神经元的作用机制.方法:采用免疫组织化学技术观察大鼠基底前脑Nestin阳性神经元雌激素α受体的表达.结果:大鼠基底前脑有20%-40%的Nestin阳性神经元表达雌激素α受体.结论:雌激素可通过雌激素α受体对大鼠基底前脑Nestin阳性神经元进行调节.4.外文期刊Liu C.Chen B.Zhu J.Zhang R.Yao F.Jin F.Xu H.Lu P Clinical implications for nestin proteinexpression in breast cancer.Recently, it was observed that nestin is preferentially expressed in basal/myoepithelial cells of the mammary gland, and thatthis intermediate filament may be used as a myoepithelial marker. However, the clinical and prognostic implications of nestin as a marker for breast cancer are still unclear. We examined mastectomy specimens from 150 breast cancers and matching, adjacent non-cancerous tissues using immunohistochemistry and western blotting. Overall, triple-negative breast cancers - that is, breast cancers that do not express estrogen receptors (ER), progesterone receptors (PR), or human epidermal growth factor receptor 2 (HER2/neu) -had higher expression rates for nestin than the other breast cancers (57.14%vs 9.30%; P < 0.001). In triple-negative breast cancers, significantly increased nestin expression rates were observed in patients with lymph node metastasis compared with those without node metastasis (25.00%vs 76.92%; P = 0.032). A similar phenomenon was observed for invasive ductal carcinomas compared with ductal carcinoma in situ (16.67%vs 73.33%; P = 0.046). Nestin expression was also found to be significantly related to ER, PR, andP53 expression (P < 0.05). Nestin expression was associated with both shorter breast cancer-specific survival and poor relapse-free survival in the lymph node-positive group (P = 0.028 and P = 0.012 respectively). After Cox regression was carried out, nestin was not shown to be an independent prognostic factor for breast cancer. These findings substantiate the possibility of using nestin as a marker for triple-negative breast cancer. Triple-negative breast cancer progression is associated with nestin; however, the underlying mechanisms of this relationship require further investigation.5.外文期刊Alliot.F.Rutin.J.Leenen.PJ.Pessac.B Pericytes and periendothelial cells of brainparenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin.Within the parenchyma of the CNS, the endothelium of all vessels is surrounded by a layer of cells, pericytes in capillaries and periendothelial or intima smooth muscle cells in other vessels. The origin of these cell types, their relationship, and their role are unclear. However, it has been recently shown that genetically engineered mice that lack pericytes develop microaneurysms at late gestation and die before birth (Lindahl et al. [1997] Science 277:242-245). The goal of this study was to identify in situ molecular markers that would be common to pericytes and periendothelial cells of adult mouse brain. Immunocytochemistry experiments werecarried out at the optical and electron-microscopic levels on mouse brain sections with antibodies specific for aminopeptidase N, aminopeptidase A, and the intermediate filament nestin. The results of our experiments show that in all brain parenchyma vessels of all sizes, pericytes and periendothelial cells are immunoreactive for aminopeptidase N, essentially at the plasma membrane level, and are also labeled by nestin specific antibodies, which decorate typical intermediate filaments. In addition, brain pericytes and periendothelial cells are also immunoreactive to monoclonal antibodies to aminopeptidase A. In contrast, pericytes andperiendothelial cells do not express microglial markers. Taken together these data show that pericytes and periendothelial intima smooth muscle cells share common markers, suggesting a common origin or function, and are distinct from microglia. Copyright 1999 Wiley-Liss, Inc.6.学位论文周立兵性腺摘除对大鼠基底前脑Nestin阳性神经元ERα表达的影响2003receptor,ER),雌激素与其受体结合能够调节胆碱能神经元的功能.雌激素水平的高低直接影响学习记忆过程.该实验室研究表明,卵巢摘除后MS-DBB复合体Nestin阳性神经元数量明显下降,雌激素替代可使以上区域Nestin阳性神经元数量恢复至正常,提示Nestin阳性神经元可能含有ER.为此,该研究进行如下实验:(1)观察Nestin阳性神经元是否表达雌激素受体以及此受体在Nestin阳性神经元表达的性别差异;(2)观察性腺摘除后基底前脑Nestin阳性神经元、ERα阳性细胞及ERα在Nestin阳性神经元的表达是否受影响.该研究的结果将为进一步探讨Nestin阳性神经元的功能特性提供新的资料和线索.7.外文期刊Karie L. Deck er.Courtney J. Conway Effects of an Unseasona ble Snowstorm on Red -FacedWar bler Nestin g SuccessEarlier initiation of nests by breeding birds may reflect an adaptive response to changes in food availability or warming of spring temperatures, but the consequences of initiating nests too early may be severe, particularly at high elevations. A rare snowstorm in late May 2008 resulted in nest abandonment by 68% of Red-faced Warblers (Cardellina rubrifrons) breeding in a high-elevation riparian ecosystem of southeastern Arizona. In addition, climate data from our study site from 1950 to 2008 revealedhigher-than-average springtime temperatures during the past 10 years. If birds respond to this increase in springtime temperatures by nesting earlier their vulnerability to spring snowstorms may increase.8.期刊论文陈二涛.潘栋超.冯东福.毕永延.汪洋.朱志安.CHEN Er-tao.PAN Dong-chao.FENG Dong-fu.BI Yong-yan.WANG Yang.ZHU Zhi-an基质细胞衍生因子-1诱导神经干细胞靶向性迁移的实验研究-中华神经医学杂志2010,9(6)目的 观察基质细胞衍生因子-1(SDF-1)对神经干细胞(NSCs)迁移的影响.方法 由胚胎大鼠脑组织获取NSCs并进行传代培养和分化鉴定,使用流式细胞术检测NSCs纯度及SDF-1特异性受体CXCR4的表达情况,之后利用Blind-Well小室体外迁移体系观察不同浓度的SDF-1(0 ng/L、1 ng/mL、10 ng/mL、50ng/mL、100 ng/mL、500 ng/mL和1000 ng/mL)对NSCs迁移数量的影响,并使用μ-载片观察SDF-1对NSCs迁移距离的影响.结果成功分离培养得到了能够在体外不断分裂增殖、具有多向分化潜能的NSCs,连续传3代后绝大部分细胞nestin表达阳性,nestin和CXCR4的共表达率达到80%左右.细胞趋化实验结果表明,SDF-1对NSCs有较强的趋化作用,随着SDF-1浓度的升高,发生迁移的细胞数量也随之增加,并于SDF-1浓度为500ng/mL时达到最高峰[(256.79±38.27)个细胞/每高倍视野].在μ-载片细胞生长通道两侧的SDF-1浓度梯度(500ng/mL→0ng/mL)作用下,由细胞克隆球迁出的细胞呈不对称分布,通常有更多的迁出细胞分布于趋化因子浓度较高的一侧,且该侧细胞的最大迁移距离也比对侧远.结论 SDF-1与其特异性受体CXCR4相巨作用能够诱导NSCs发生靶向性迁移.9.期刊论文王留东.赵二义.贾延劼.WANG Liu-dong.ZHAO Er-yi.JIA Yan-jie小凹蛋白-1在大鼠骨髓间质干细胞分化为神经元样细胞中的作用-中国实用医刊2010,37(11)目的 应用RNA干扰(RNA interference)技术,抑制沉默小凹蛋白-1(caveolin-1)基因的表达,观察小凹蛋白-1在骨髓间充质干细胞(MSCs)分化为神经元样细胞的作用.方法 构建大鼠caveolin-1 siRNA(small interfering RNA),然后传染大鼠MSCs,诱导分化并在倒置显微镜下进行形态学观察,用MTT比色法检测细胞存活率,采用免疫细胞化学染色法检测Nestin、 NSE(神经元标记物) 、GFAP(神经胶质细胞标记物)的表达,设诱导未传染组和传染对照组作为对照研究.结果 用caveolin-1 siRNA传染MSCs后,caveolin-1基因表达消失,传染后24 h和3 d MSCs细胞存活率下降,与诱导未传染组和传染对照组比较差异有统计学意义(P<0.01),传染caveolin-1 siRNA的MSCs向神经元样细胞分化效率显著提高,诱导24 h后NSE阳性细胞率为(75.5±2.5)%,6 d后达(81.6±3.5)%,且未见GFAP的表达,与诱导未传染组和传染对照组比较差异有统计学意义(P<0.01).结论 caveolin-1 siRNA抑制caveolin-1基因的表达,可提高MSCs向神经元样细胞的分化效率.10.学位论文徐嘉雯基底细胞样乳腺癌及三阴性乳腺癌的临床病理特点及分子标记物分析2008【目的】探讨基底细胞样乳腺癌与三阴性乳腺癌的临床病理特征及分子标记物表达的特点,为临床病理诊断和乳腺癌患者的个体化治疗提供依据。
Mouse Scfv Module

READ ME FIRST•This is an electronic version, produced inAdobe Acrobat, of the instructionssupplied with the product.•On most monitors, use of the file issimplest at 115-150% magnification inAcrobat. This magnification should makethe print size easy to read and permitorientation through the instructions.•The page numbering of the Acrobat filedoes not match the page numbering ofthe instruction booklet, since twoinstruction booklet pages have beenassembled onto one Acrobat page.Therefore, references to page numberswithin the instructions are referring to thepage numbers within the instructions andnot to the numbers of the Acrobat pages.A M E R S H A MB I O SC I E N C E S Mouse ScFv Module/Recombinant PhageAntibody System INSTRUCTIONS The Recombinant Phage Antibody System is designed to clone mouse antibody genes and to express and detect functional antibodies. It is based on a phage-display system in which fragments of antibodies are expressed as fusions with phage proteins and displayed on the phage surface. It is designed in a flexible modular format with three parts: Mouse ScFv Module; Expression Module; and Detection Module.This booklet provides the protocols for the Mouse ScFv Module. This module is designed to produce an ScFv gene fragment ready for ligation into the pCANTAB 5 E vector supplied in the Expression Module.A complete overview of the system is provided separately. Instructions for the other two modules of the system are provided with those products.The technology was developed in collaboration with Cambridge Antibody Technology Ltd., UK. See page 3 for important licensing information.XY-039-00-07Rev. 4CONTENTSPatent and Licensing Information3 Components4 Protocols6 ProceduresA:First-Strand cDNA Synthesis 13B:Primary PCR Amplification14C:Purification of Primary PCR Products15D:Gel Quantitation of Purified Product17E:Assembly and Fill-In Reactions19F:Second PCR Amplification and Purification20G:Gel Quantitation of Purified ScFv 23H:Restriction Digestion and Purification25 Appendices1:Additional Materials Required272:mRNA Isolation from Hybridomas293:mRNA Isolation from Spleen334:Reamplification of Primary PCR Products355:Reamplification of ScFv Fragments37 Troubleshooting Guide38 Function Testing, Storage42 References, Ordering Information42Copyright © 1995 by Amersham Biosciences Inc.,Molecular Biology Reagents Division2PATENT AND LICENSE INFORMATIONBefore using this product, carefully read the Patent and License Information below.By using this product, you indicate your acceptance of the conditions described below.A license for research use only of the technology which covers the phagemid vector, antibody primers, and amplification methods related to the Recombinant PhageAntibody System, and the antibodies or molecules derived therefrom, is granted by Cambridge Antibody Technology to the end-user to use the technology, provided the Module has been purchased from Amersham Biosciences. If you are in doubt as to whether your use of antibodies or molecules derived from them would require a commercial license, please discuss the matter with Cambridge Antibody Technology (see the instruction booklet for the Expression Module).The Amersham Biosciences Recombinant Phage Antibody System/Mouse ScFv Module product is designed and intended for research use only, not for diagnostic or medical use. The technology covering the construction of single-chain F-variable (ScFv) recombinant antigen-binding molecules is covered by U.S. Patent No. 4,946,778 and other domestic and foreign patent rights, owned by Enzon Inc., and licensed by Amersham Biosciences. The purchase of an Amersham Biosciences Recombinant Phage Antibody System/Mouse ScFv Module includes a fully paid-up, limited license under such patent rights to use these products for research use only. For this purpose, the license “for research use only”means research activities including screening, selection and identification of ScFv molecules. No license is granted for any other uses, such as (without limitation) (1)pre-clinical toxicological studies with ScFv or any research or commercial activities beyond such toxicological studies; (2) diagnosis or detection of disease or other health-related conditions in humans, animals or plants using ScFvs; or (3) therapeutic applications of any type using ScFvs. No other license is granted, expressly, impliedlyor by estoppel. For information concerning the availability of additional licenses toutilize single-chain antigen-binding molecules contact Enzon Inc., 40 Cragwood Road, South Plainfield, New Jersey 07080.COMPONENTSPrimed First-StrandFPLC pure ®Cloned Murine Reverse Reaction Mix :Transcriptase, random hexadeoxyribo-nucleotides [pd(N)6], RNAguard ®, RNase/DNase-free BSA, dATP , dCTP , dGTP anddCTP in aqueous buffer .Control mRNA :Mouse hybridoma mRNA in RNase-freewater .DTT Solution :200 mM DTT solution.RNase-free Water :Treated with diethyl pyrocarbonate (DEPC).Heavy Primer 1:5' heavy chain primer in water (also referredto as upstream primer).Heavy Primer 2:3' heavy chain primer in water (also referredto as downstream primer).Light Primer Mix :Mixture of 10 variable light chain primers(both 5' and 3') in water .Linker-Primer Mix :Equimolar mixture of 3' heavy and 5' lightlinker primers in water (sufficient for25 assembly reactions).RS Primer Mix :Mixture of 5' heavy chain primer with Sfi Isite and 3' light chain primer with Not I sitein water (sufficient for 25 amplificationreactions).V H Marker :pUC18/V H , Eco R I/Xba I digested.ScFv Marker :pUC18/A10B,Sfi I/Not I digested.4Sephacryl ®S-400 HR*:Sephacryl ®S-400 HR suspended in distilled water and 20% ethanol.MicroSpin ™Columns (25):Polypropylene minicolumns, each fitted with a 10 µm frit.*Resin is supplied frozen. Thaw completely before use.All components provided as part of the Mouse ScFv Module are inboldface type ; additional materials and reagents are described inAppendix 1, page 27.Break hereNote:Before using a MicroSpin Column, snapoff the bottom closure.PROTOCOLSThe Mouse ScFv Module is designed to produce an ScFv gene fragmentready for ligation into the pCANTAB 5 E vector supplied in the Expres-sion Module. For a detailed description of the cloning procedure, refer tothe Overview booklet. The flow chart on pages 8-10 summarizes the procedure in this Module.mRNA PurificationmRNA can be isolated from either hybridoma or spleen cells according tothe protocols outlined in Appendices 2 and 3, pages 29 and 33.Special precautions must be observed to avoid degradation of mRNA by RNases. Reagents and plastic- or glassware that contact the RNA sample should first be treated with diethyl pyrocarbonate (DEPC) to inactivateany contaminating RNases and then autoclaved according to standard procedures (1). Gloves must be worn to avoid sample contamination with nucleases shed from the skin.Highly pure mRNA is required as starting material for this kit. The quali-ty of mRNA is very important in generating recombinant antibodies. The QuickPrep® mRNA Purification Kit from Amersham Biosciences is highly recommended for purifying mRNA from hybridoma cells (see Appendix 2, page 29, for details on its use). Any alternate scheme should yield mRNA freeof extraneous DNA, rRNA or tRNA since these contaminants may inter-fere with subsequent reactions. At least one round of affinity chromatog-raphy using oligo(dT)-cellulose should be included in all protocols. Regardless of the method chosen, the mRNA should be ethanol-precipitated and dissolved in 20 µl of RNase-free water prior to use in thefirst-strand cDNA synthesis reaction (Procedure A). The RNA Extraction6Kit, followed by the mRNA Purification Kit (both from Amersham Biosciences), is recommended for purifying mRNA from spleen (see Appendix 3, page 33,for details).Control Mouse mRNAThe Control mRNA included in the Mouse ScFv Module is derived from hybridoma cells. The hybridoma cell line produces monoclonal antibody which reacts with polyclonal rabbit IgG. Control mRNA has been testedto ensure that it generates primary PCR products of the expected size(~325 bp for antibody light chain and ~340 bp for antibody heavy chain DNA). Sufficient Control mRNA is supplied for one heavy and one light chain reaction, using 20 µl of reagent for each reaction. This mRNA canbe taken through the entire RPAS as a system control.Amplification Reactions*Reagents for the amplification reactions are available from Perkin-Elmer Cetus.The primers used in the primary PCR amplification of antibody first-strand cDNA will generate suitable quantities of antibody heavy(~340 bp) and light (~325 bp) chain DNA for cloning. During the(continued on page 11)*The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202 owned byHoffmann-La Roche Inc. Use of the PCR process requires a license. Nothing in thisset of instructions should be construed as an authorization or implicit license topractice PCR under any patents held by Hoffmann-La Roche Inc.8Reverse Transcriptase + pd(N)6AAAA TT T T C (continued on page 9)AmpliTaq ® + dNTPs30 cyclesAAAA TTTT V V C 2C 1C3(continued on page 10)Amplification 1 (cont.)LH10I DigestV L V H Linker Sfi I Not I++I Digest+(To Expression Module)V L V H 30 cyclesAmpliTaq ® + dNTPs L Sfi I Not I H assembly reaction, the heavy and light chain DNA fragments are joined with a specially constructed linker DNA. The assembled ScFv product is subsequently amplified with primers which attach Sfi I and Not I restriction sites.The assembly reaction can be problematic. For this reaction to be suc-cessful, purified heavy and light chain product must be added in approx-imately equimolar concentrations. Assembly will proceed optimally if approximately 50 ng of each product (heavy and light chain) are added to this reaction, along with the recommended amount of linker primers. Following assembly , the amplified product (~750 bp) is digested with Sfi I and Not I for subsequent cloning into the pCANT AB 5 E phagemid vector .Agarose Gel PurificationThe heavy and light chain DNA fragments from the primary PCR (Procedure B, page 14) must be separated from other components on an agarose gel, extracted and purified. A quick and easy method using the MicroSpin ™Columns * provided with the kit can be used for this step. Other suitable methods such as Sephaglas ™BandPrep Kit from Amersham Biosciences may also be used.–Note: TAE buffer , rather than TBE buffer , should be used for agarose gelelectrophoresis prior to band isolation.*MicroSpin is a trademark of Lida Manufacturing Corp.11Quantitation of Purified ProductsFor the assembly reaction to proceed optimally, approximately 50 ng each of purified heavy and light chain product must be used. It is impor-tant that the amounts of heavy and light chain in the reaction are approximately equimolar. To determine the appropriate amount of each to use, aliquots of the heavy and light chain products are analyzed on an agarose gel in parallel with a known quantity of DNA marker.The efficiency of the restriction digestion of the assembled ScFv frag-ments depends upon the number of units of enzyme used for a given quantity of product. T o determine the appropriate amount of ScFv to add to the restriction digests, an aliquot of the purified ScFv is run on an agarose gel in parallel with a known quantity of DNA marker.Sfi I and Not I Digestion of the ScFv GeneThe ScFv gene is digested with Sfi I followed by Not I and is purified in preparation for ligation to the predigested pCANTAB 5 E provided in the Expression Module.12Procedure A:First-Strand cDNA SynthesisM-MuL V reverse transcriptase, supplied as a component of the Primed First-Strand Mix, is used in the first-strand cDNA reaction to generate cDNA from the mRNA (see Appendix 2 or 3 for mRNA preparation). Prior to first-strand cDNA synthesis, the mRNA should be ethanol-precipitated, resuspended in 20 µl of RNase-free water and heated to remove any secondary structure which could hinder the reaction.–Note: To produce an ScFv molecule, you will need to do two first-strand cDNA synthesis reactions: one for the heavy chain and one for the light chain.•Heat the mRNA at 65°C for 10 minutes and cool immediately on ice. Start the first-strand cDNA reaction (below) within 2 minutes after placing on ice.•For each sample, prepare the following reaction in duplicate in 500 µl microcentrifuge tubes. Label one tube as light chain and the second tube as heavy chain.mRNA*XµlRNase-free Water XµlPrimed First-Strand Mix11µlDTT Solution1µlTotal Volume33µl*For spleen mRNA, use 200 ng to 1 µg per reaction. For hybridoma mRNA, use 1/4 of the eluate when starting from 5 x 106viable cells. For the Control mRNA included in the kit, use 20 µl per reaction and 1 µl of RNase-free water.•Incubate for 1 hour at 37°C.Procedure B:Primary PCR AmplificationThe first-strand antibody cDNA is used as a template for PCR ampli-fication to generate suitable quantities of antibody heavy (~340 bp) and light (~325 bp) chain DNA for cloning.•Add the reagents shown below to the appropriate tubes containing the first-strand reactions from Procedure A:Light Chain PCRFirst-strand reaction33µlLight Primer Mix2µlSterile distilled water64µlTotal Volume99µlHeavy Chain PCRFirst-strand reaction33µlHeavy Primer 12µlHeavy Primer 22µlSterile distilled water62µlTotal Volume99µl•Mix gently with a micropipettor and spin briefly. Overlay each reaction with 100 µl of mineral oil. Place the tubes in a thermocycler and heat at 95°C for 5 minutes. Add 1 µl of AmpliTaq®DNApolymerase (Perkin-Elmer Cetus) to each reaction beneath themineral oil, using separate pipet tips for each addition.•Return the tubes to the thermocycler and immediately run Program 1(30 cycles: 94°C for 1 minute; 55°C for 2 minutes; 72°C for2 minutes).14Procedure C:Purification of Primary PCR Products Prior to performing the assembly reaction, the amplified heavy and light chain PCR fragments must be isolated from the other reaction compo-nents by electrophoresis on a preparative agarose gel. Purification of the DNA from an agarose gel can be done quickly and easily using the empty MicroSpin Columns provided in the kit, as described below. Alterna-tively, commercially available purification systems such as Sephaglas™BandPrep Kit (27-9285-01) may be used.•Immediately prior to use, prepare a 1.5% agarose gel (~1 cm thick,0.5X TAE buffer) with wells sufficient to accommodate 50 µl samples.–Note: Use TAE buffer, rather than TBE buffer, for gel electrophoresis.•Remove ~85 µl of the amplification reactions (excluding mineral oil) and transfer to separate microcentrifuge tubes. Store the remainder of the mixes at -20°C.•Add 15 µl of 6X loading dye (see Appendix 1, page 27) to each sample and mix well. Load two 50 µl aliquots of the reaction mix into adjacent wells. Electrophorese until the bromophenol blue dye hasmigrated approximately two-thirds the length of the gel.–Note: Use dye containing bromophenol blue and not xylene cyanole. If xylene cyanole is used in the dye, it may obscure the heavy and light chains.•Using a scalpel, excise the 340 bp (heavy chain) and 325 bp (light chain) DNA bands, and carefully trim away any excess agarose. Use a different scalpel for each chain to prevent contamination.–Note: Some hybridomas may produce more than one heavy or light chain antibody molecule, thus giving rise to multiple DNA products from the primary PCR. For example, NS-1 myeloma cells, which are used as fusion partners togenerate some hybridomas, produce an aberrant light chain. If a series of co-migrating bands is seen, isolate the DNA from each band and pool the isolates.Recombinant phage antibodies specific for the antigen can then be selected at a later stage when the phage antibodies are affinity-selected against antigen.•Lightly blot each piece of agarose briefly with a paper towel and slice it into tiny pieces with a scalpel. Place all the pieces from the heavychain and all the pieces from the light chain into two separate empty MicroSpin Columns. These columns should not contain any resin!•Break off the bottom of the column and discard the bottom piece.Place each column in a 1.5 ml microcentrifuge tube and centrifuge at 735 x g* for 2 minutes. The resulting eluate is the purified DNA,which can be used directly in the assembly reaction (Procedure E,page 19).–Note: The agarose pieces and the eluted solution can both be examined briefly under UV light to determine the efficiency of the purification. Some ethidiumbromide-stained material will remain in the agarose pieces; in fact, the stainingintensity of the agarose pieces and the eluted solution may often be similar.*Note: If you do not have a microcentrifuge, you may use a clinical centrifuge set at a speed corresponding to 735 x g (please see below). Place the MicroSpin Column in a 1.5 ml microcentrifuge tube for support and set the support tube in a 100 x 13 mm test tube. Place the entire support system, including shields, in a clinical centrifuge (e.g. IEC model HN-SII) and proceed as described above.Centrifugation conditions: Before using a MicroSpin Column, it is important to calculate the speed at which the column should be centrifuged. For a force of 735 x g, the appropriate speed can be calculated from the following formula:rpm = (1000)( 657/r ),where r = radius in mm measured from center of spindle to bottom of rotor bucket and rpm = revolutions per minute. For example, with a rotor having a radius of 110 mm, the appropriate speed would be 2444 rpm.16Procedure D:Gel Quantitation of Purified Product The success of the assembly and fill-in reaction (Procedure E) is dependent on the molar concentrations of both heavy and light chain PCR products. Agarose gel electrophoresis of aliquots of the purified heavy and light chain products alongside a known amount of V H Marker provides a visual estimate of the relative amounts of the fragments based on their band intensities in an ethidium bromide-stained gel. Fifty ng of each chain should be added to the assembly reaction for optimal results.•Prepare a small, thin (~0.5-0.75 cm thick) 0.5X TAE agarose gel (1.5% agarose) with wells ~3 mm in width. If desired, add ethidium bromide to the gel and running buffer to a final concentration of 0.5 µg/ml each.–Note: Thin gels must be used since the quantities of DNA loaded onto the gel will be difficult to visualize if thicker gels are used.•T o two separate 1.5 ml microcentrifuge tubes, add 1/20 and 1/40 of the total amount of purified heavy chain. If necessary, adjust the volume to5 µl with TE buffer. T o each tube, add 1 µl of 6X loading dye (seeAppendix 1).•T o two separate 1.5 ml microcentrifuge tubes, add 1/20 and 1/40 of the total amount of purified light chain. If necessary, adjust the volume to5 µl with TE buffer. T o each tube, add 1 µl of loading dye.•T o prepare the marker, add 2.5 µl of the V H Marker and 2.5 µl of TE buffer to 1 µl of loading dye in a microcentrifuge tube. Also preparea second tube containing 5 µl of V H Marker and 1 µl of loading dye.–Note: At this point you should have two tubes for the heavy chain, two tubes for the light chain, and two tubes for the V H Marker, for a total of six tubes.•Load all six samples into separate wells of the agarose gel and electro-phorese until the bromophenol blue dye has migrated approximately two-thirds the length of the gel.•If ethidium bromide was not added to the gel and running buffer, stain the gel with ethidium bromide. Photograph under UV light. It may be necessary to over-expose the picture to visualize the marker and theheavy and light chain products.•Compare the intensities of the heavy and light chain products with the V H Marker band which is slightly larger (~370 bp) than the size of the heavy and light chain products. This marker band contains approxi-mately 12.5 ng in the 2.5 µl aliquot and approximately 25 ng in the5 µl aliquot. Note that the V H Marker also contains a higher molecularweight band.•Estimate the volume of purified heavy chain product that corresponds to 50 ng. Estimate the volume of purified light chain product that cor-responds to 50 ng. If the combined required volume of heavy and light chain product is <32.5 µl, proceed to Procedure E, page 19.•If the combined required volume of heavy and light chain product is >32.5 µl, precipitate the required volumes of the two products in sepa-rate microcentrifuge tubes (see below) or reamplify the primary PCR products (see Appendix 4, page 35).For ethanol precipitation: T o each sample, add 1/10 volume of 3 Mpotassium acetate (pH ~5) and 2.5 volumes of cold 100% ethanol.Precipitate for 20 minutes on Dry Ice or for 1 hour at -20°C. Centrifuge each sample for 10 minutes at full speed in a microcentrifuge(~14,000 x g), discard the supernatants, and wash the pellets with cold 70% ethanol. Repeat the centrifugation step once more. Dry the DNA pellets (which will probably not be visible) completely. Resuspend each pellet in 15 µl of TE buffer. Use the appropriate volumes (correspond-ing to 50 ng each) in the assembly reaction.18Procedure E:Assembly and Fill-In ReactionsIn the assembly reaction, the antibody heavy and light chain DNA are joined into a single chain with linker DNA. When the linkers anneal to the heavy and light chain DNA, they prime a fill-in reaction in the presence of AmpliTaq®DNA Polymerase. For this reaction to proceed efficiently, approximately 50 ng of heavy chain and 50 ng of light chain should be added to the reaction.•Add the following components to a 500 µl microcentrifuge tube: Heavy chain product (50 ng)XµlLight chain product (50 ng)XµlLinker-Primer Mix 4µl10X PCR Buffer5µldNTP Mix (20 mM of each dNTP) 2.5µl25 mM MgCl25µlAmpliTaq®DNA Polymerase0.5-1.0µl (2.5-5 U)Sterile distilled water to 50.0µlTotal Volume50.0µl•Mix and spin briefly. Overlay with 50 µl of mineral oil.•Place the tube in a thermocycler and run Program 2 (7 cycles, as follows: 94°C for 1 minute; 63°C for 4 minutes).–Note: If a dNTP mix of lower concentration is used, the volume added must be scaled up proportionally.20Procedure F:Second PCR Amplificationand PurificationIn the second PCR, the assembled ScFv DNA is amplified and restriction sites are added. These restriction sites are used to clone the DNA into the phagemid vector . The RS Primers (Restriction Site Primers) contain either Sfi I or Not I restriction sites. They anneal to the 5' end of the heavy chain (Sfi I restriction site) or the 3' end of the light chain (Not I restriction site).PCR•Prepare a bulk primer mix by adding the following components to a 500 µl microcentrifuge tube. For each PCR, use:AmpliTaq ®DNA Polymerase 0.5-1µl (2.5-5 U)10X PCR Buffer 5µldNTP Mix (20 mM of each dNTP)1µlRS Primer Mix 4µlSterile distilled water 39µlTotal Volume 50µl•Mix briefly and add the bulk primer mix to the assembly reaction (from Procedure E) beneath the mineral oil. Overlay the reaction with an additional 50 µl of mineral oil.•Place the tube in a thermocycler and run Program 1 (30 cycles: 94°C for 1 minute; 55°C for 2 minutes; 72°C for 2 minutes).•Analyze 5 µl of each reaction on a 1.5% agarose gel. A predominant band ~750 bp in size should be present. Some heavy and light chain monomers may also be visible.MicroSpin Column PreparationSee Appendix 1, page 27 for preparation of TE Buffer .MicroSpin Columns are used to separate the 750 bp assembled ScFv product from the components of the PCR mix, which may interfere with subsequent restriction digestions.•Resuspend the thawed Sephacryl ®S-400 HR resin completely by gentle shaking and immediately add 750 µl of the suspension to a MicroSpin Column . Place the column in a 1.5 ml microcentrifuge tube and spin at 735 x g for 1 minute (DO NOT PULSE!). T o calculate the g force and for alternatives to variable-speed centrifuges, see below.Centrifugation conditions : Before using a spun column, it is important to calculate the speed at which the column should be centrifuged. For a force of 735 x g, the appropriate speed can be calculated from the following formula:RCF = 735 = (1.12)(r)(rpm/1000)2, where RCF = relative centrifugal force;r = radius in mm measured from center of spindle to bottom of rotor bucket; and rpm = revolutions per minute. The above equation resolves to: rpm = (1000)( 657/r ).For example, with a rotor having a radius of 110 mm, the appropriate speed would be 2444 rpm.–Note: As an alternative to spinning in a variable-speed microcentrifuge, a clinical (tabletop) centrifuge with a swinging-bucket rotor may be used. Tospin, place a 12 x 75 mm disposable glass centrifuge tube inside an adaptor to hold the MicroSpin Column. Set the speed so that an rpm setting correspond-ing to 735 x g will be achieved. Start the centrifuge, but begin timing onlywhen the rotor has reached the set speed. Spin for 1 minute at this speed, then apply the brake until the rotor stops. Wash the column as in the standardprotocol. Just prior to sample application, place a clean 1.5 ml microcentrifuge tube inside the glass support tube and return the column to the microcentrifuge tube. Spin as outlined above.•Empty the microcentrifuge tube and replace the column inside the tube. Add 200 µl of TE buffer to the top of the resin and spin asoutlined above.•Repeat this step one time.Purification•Transfer the column to a sterile 1.5 ml microcentrifuge tube. Care-fully apply the entire volume (~100 µl) of the second PCR product (excluding mineral oil) to the top of the compacted column resin and spin at 735 x g for 2 minutes.•Discard the column. The effluent in the microcentrifuge tube contains the purified assembled ScFv DNA which is ready for quantitation on an agarose gel alongside the ScFv Marker.22Procedure G:Gel Quantitation of Purified ScFv PCR products are notoriously difficult to digest efficiently with restric-tion enzymes. The number of units of restriction enzyme per unit quan-tity of PCR product should be optimized to promote efficient cleavage of the PCR product. The conditions for the restriction enzyme digests of the assembled ScFv (Procedure H) are set up so that 0.25-1 µg of assem-bled product will be digested efficiently. To determine the volume of purified ScFv product to add to the restriction digests, a gel quantitation step is performed. A portion of the assembled product is run on an agarose gel containing ethidium bromide alongside a known amount of ScFv Marker, then visualized under UV light. The intensity of the assembled product is then compared with the intensity of the band of the ScFv Marker that corresponds to the size of the assembled product (a second band of higher molecular weight will also be present in the marker), and an estimate is made of the amount of DNA in the portion of assembled product loaded onto the gel. Based on this visual estimate, the appropriate amount of assembled product can be digested with Sfi I and Not I.•Prepare a small, thin (~0.5-0.75 cm thick) 0.5X T AE agarose gel(1.5% agarose) with wells ~3 mm in width. If desired, add ethidiumbromide to the gel and running buffer to a final concentration of0.5 µg/ml each.–Note: Thin gels must be used since the quantities of DNA loaded onto the gel will be difficult to visualize if thicker gels are used.•To a 1.5 ml microcentrifuge tube, add 2 µl of purified assembled product, 3 µl of TE buffer, and 1 µl of 6X loading dye (see Appendix1). To a second tube, add 4 µl of purified assembled product, 1 µl ofTE buffer, and 1 µl of loading dye.。
三阴乳腺癌的研究进展(1)

垄宝医堂盘查!Q塑生箜丝鲞筮!塑三阴乳腺癌的研究进展辽宁中医药大学(110032)芦洪波沈阳军区总医院肿瘤科谢晓冬(审校)乳腺癌是一种高度异质性的恶性肿瘤,不同类型的乳腺癌在生物学行为、临床病理特征及治疗效果等方面都存在很大差异。
1983年全国乳腺癌病理协作会议将乳腺癌分为非浸润性癌、早期浸润性癌、浸润性特殊癌和浸润性非特殊癌4大类。
随着肿瘤学科的发展,传统的肿瘤病理学分型方法面临着巨大挑战。
近年来,越来越多能够反映癌症治疗靶点和预后的标记物浮出水面。
其中,雌激素受体(ER)、孕激素受体(PR)和人表皮生长因子受体2(HER2)已成为乳腺癌患者中免疫组化标记物常规检查项目,针对ER、PR、HER2的靶向治疗目前已取得很大进展。
三阴乳腺癌为ER、PR、HER2均阴性的乳腺癌,是一类具有特殊生物学特征和临床病理特征的乳腺癌亚型。
由于无内分泌治疗及针对HER2的分子靶向治疗的选择,化疗成为此型乳腺癌患者的主要内科治疗途径。
本文将从三阴乳腺癌的生物学特征、临床病理特征和治疗策略三个方面进行综述。
1生物学特征三阴乳腺癌是一类具有特殊生物学特征的乳腺癌亚型。
Rakha等¨1研究表明,三阴乳腺癌的生物学特征有:肿瘤体积大、病理类型多为导管癌、组织学分级较高、肿瘤细胞多数表达CK5/6和基底细胞角蛋白(CKl7)、P-钙黏蛋白(P—cadherin)、EGFR和p53,较少表达雄激素受体(AR)、E—cadherin、细胞周期蛋白D(cyclinD)。
已有的研究表明,通过病理特征分析,三阴乳腺癌组织学分级和细胞增殖比例均较高,干细胞因子受体c(c一Ⅺt)多为阳性Ⅲ】。
三阴乳腺癌与基底样乳腺癌具有一定相关性。
基底样乳腺癌是采用基因芯片技术对乳腺癌进行基因表达谱分析得出的乳腺癌亚型。
由于基因芯片成本高,不可能在临床工作中常规应用。
在此条件下,寻找能够替代基因表达谱分析的免疫组化标记物是最好的方法之一。
基底样乳腺癌与三阴乳腺癌相关联,因为两者在mRNA和蛋白表达水平上十分相似。
顺铂诱导三阴性乳腺癌4T1耐药小鼠模型的建立

顺铂诱导三阴性乳腺癌4T1耐药小鼠模型的建立盛佳钰;陈红风【摘要】Objective To establish a cisplatin-resistant 4T1 mouse model of triple negative breast cancer .Meth-ods A drug resistant mice model was established with cisplatin ( DDP ) induction and in-vivo/in-vitro tumorigenic ap-proach.Its resistance characteristics were identified by MTT assay .Changes of drug resistance gene ( MDR1, BCRP, MMP7, GST-π) and protein ( P-gp, BCRP, MMP7) expression, and phosphorate-Akt and total-Akt protein expression were evaluated by real-time PCR, immunohistochemistry and western blot method , respectively.Small animal live imaging technology was applied to detect tumorgrowth .Results Resistance fold (RF) of cisplatin-resistant 4T1 mouse model was 12.84.The expression of MDR1, BCRP, MMP 7, GST-πmRNA and P-gp, BCRP, MMP 7 proteins in the resistant mice were higher than that in the non-resistant mice .The result of western blot showed that a statistically higher expression of p-Akt in resistant mice than that in non-resistant mice at protein levels (P<0.01).No significant difference of tumor growth rate was observed between non-resistant and resistant mice ( P>0.05 ) .Given same dose of DDP , resistant mice showed lower sensitivity than non-resistant mice significantly (P<0.01).Conclusions We have successfully established a cis-platin-resistant triple negative breast cancer model in mice , which provides a new platform for further study on chemoresis-tant reversal strategy and individualized clinical treatment of this disease .%目的建立一种耐药性稳定的三阴性乳腺癌4 T1耐药小鼠模型,为研究体内肿瘤耐药机制和逆转药物的筛选奠定实验基础. 方法采用顺铂( DDP)低剂量诱导及体内外交叉致瘤结合的方法建立三阴性乳腺癌耐药小鼠模型;MTT法检测细胞耐药特性;实时荧光定量PCR法分析耐药相关基因MDR1、BCRP、MMP7及 GST-π表达差异;免疫组化分析耐药相关蛋白P-gp、BCRP、MMP7表达差异;蛋白印迹法检测磷酸化Akt(phosphorate-Akt, p-Akt)和总Akt( total-Akt,t-Akt)蛋白表达;小动物成像检测观察肿瘤生长情况. 结果 MTT显示建立的三阴性乳腺癌耐药4T1小鼠模型的耐药指数为12.84;耐药小鼠肿瘤组织中MDR1、BCRP、MMP7、GST-π基因mRNA 的表达量及P-gp、BCRP、MMP7蛋白的表达量均高于非耐药小鼠(P<0.01);Western blot显示,耐药小鼠肿瘤组织的p-Akt蛋白表达明显高于非耐药小鼠,t-Akt蛋白表达没有差异. 非耐药小鼠与耐药小鼠肿瘤组织生长速度未见明显区别(P>0.05). 分别给予这两种模型小鼠相同剂量的DDP治疗后,耐药小鼠对DDP的敏感性明显低于非耐药小鼠(P<0.01). 结论初步建立了三阴性乳腺癌耐药4T1小鼠模型,为三阴性乳腺癌临床个体化治疗及耐药逆转研究等提供了良好的实验动物平台.【期刊名称】《中国实验动物学报》【年(卷),期】2015(023)005【总页数】8页(P466-473)【关键词】三阴性乳腺癌;肿瘤抗药性;顺铂;动物模型;小鼠【作者】盛佳钰;陈红风【作者单位】上海中医药大学附属龙华医院乳腺科,上海 200032;上海中医药大学附属龙华医院乳腺科,上海 200032【正文语种】中文【中图分类】Q95-33目前认为乳腺癌是一种高度异质性肿瘤,不同的病理特点和分子特征导致其对治疗的反应和预后明显不同。
IBM Cognos Transformer V11.0 用户指南说明书

基于化学遗传的慢性应激导致抑郁症小鼠模型的构建

第42卷第3期2021年5月Vol.42No.3May2021中山大学学报(医学科学版)JOURNAL OF SUN YAT⁃SEN UNIVERSITY(MEDICAL SCIENCES)基于化学遗传的慢性应激导致抑郁症小鼠模型的构建邓艺雯,张寅航,徐兴浩,蚁焌哲,张小然,黄玮俊(中山大学中山医学院干细胞与组织工程研究中心,广东广州510080)摘要:【目的】建立基于化学遗传的慢性应激导致抑郁症的小鼠模型,为优化慢性应激诱发抑郁症的动物建模方法提供参考。
【方法】通过化学遗传的方法,对小鼠延髓腹外侧区头端(RVLM)进行持续的刺激,模拟机体对慢性应激的响应,系统观察造模和对照组小鼠的行为学、血压及主要炎症因子的变化情况。
【结果】成功建立了小鼠模型。
经腹腔向小鼠给药CNO1h后,造模组较对照组的舒张压与平均脉压显著上升。
持续给药4周后,造模组较对照组的旷场实验的水平运动总距离、中央区进入总次数、中央区滞留时间和中央区域运动总距离这四项指标均显著下降;蔗糖偏好实验提示造模组存在快感缺失;转棒实验提示造模组运动协调能力显著减弱;同时,造模组心率、血压、炎性因子IFN-γ和抑炎因子IL-10水平等也较对照组显著上调。
【结论】我们的模型在诱发抑郁症经典症状,及血压偏高、炎性因子释放紊乱等抑郁症伴随症状上均表现出较高的成功率和稳定性。
这为优化慢性应激诱发抑郁症的动物建模效率和稳定性提供了有参考价值的科学依据。
关键词:抑郁症;慢性应激;交感神经;化学遗传;延髓腹外侧区头端中图分类号:R338.2+2文献标志码:A文章编号:1672-3554(2021)03-0346-09DOI:10.13471/ki.j.sun.yat-sen.univ(med.sci).2021.0104Establishment of Chronic Stress Inducing Depressive Disorder Mouse ModelVia Chemical GeneticsDENG Yi-wen,ZHANG Yin-hang,XU Xing-hao,YI Jun-zhe,ZHANG Xiao-ran,HUANG Wei-jun(Center for Stem Cell Biology and Tissue Engineering,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou510080,China)Correspondence to:HUANG Wei-jun;E-mail:*****************Abstract:【Objective】To establish a chronic stress inducing depressive disorder mouse model via chemical genetics,that helps to optimize the animal modeling method of chronic stress-induced depressive disorder.【Methods】To continuous⁃ly stimulate the rostral ventrolateral medulla(RVLM)of mouse by chemical genetic method to mimic the response to chron⁃ic stress.The changes about behavior,blood pressure and major inflammatory factors of the animal models were corre⁃spondingly observed.【Results】The mouse model was established successfully.One hour after intraperitoneal administra⁃tion of CNO,diastolic blood pressure and mean blood pressure were significantly increased in the model group compared with the control.After four weeks of continuous administration,the total distance of movement,the total entering bouts,the distance and duration of the central area in the model group were significantly decreased compared with the control in the open field experiment.The results of sucrose preference and rotarod experiment suggested anhedonia and weakened mo⁃tor coordination ability in the model group.The heart rate,blood pressure,expressions of IFN-γand IL-10in the model group were higher than those in the control.【Conclusions】Our model developed classic symptoms of depressive disorder ef⁃ficiently and stably,as well as accompanying symptoms such as high blood pressure and mussy release of inflammatory fac⁃tors.It provides a valuable scientific basis for improving the efficiency and stability of animal modeling of chronic stress-induced depressive disorder.Key words:depressive disorder;chronic stress;sympathetic nerve;chemical genetics;RVLM[J SUN Yat⁃sen Univ(Med Sci),2021,42(3):346-354]收稿日期:2021-03-01基金项目:国家自然科学基金(81970222);广东省科技计划项目(2016B090918040,2017B020230004)作者简介:邓艺雯,硕士生,研究方向:干细胞与组织工程,E-mail:******************;黄玮俊,通信作者,副教授,博士生导师,E-mail:*****************第3期邓艺雯,等.基于化学遗传的慢性应激导致抑郁症小鼠模型的构建抑郁症(depressive disorder)是一种常见的精神类疾病,核心症状是情感低落(depressed mood)、兴趣减退或快感缺失(anhedonia)。
人二氢乳清酸脱氢酶抑制剂的潜在用途

科技风2021年4月机械化工DOE10.19392/ki.1671-7341.202112084人二氢乳清酸脱氢酶抑制剂的潜在用途叶磊冉莎任小利*张伟舒蕾刘庆香重庆化工职业学院重庆401228摘要:人二氢乳清酸脱氢酶'Human dihydroorotate dehydrogenase,hDHODH)是一种参与嘧啶从头合成途径的限速酶,迅速生长和分裂的细胞需要大量的从头合成嘧啶来维持其增长,因此,抑制hDHODH则可抑制嘧啶的从头合成,从而抑制细胞的快速增殖,研究表明,hDHODH可能是一个治疗癌症、移植排异、类风湿性关节炎、自身免疫性疾病、抗病毒等的潜在靶标。
本文主要讨论hDHODH在癌症和抗病毒领域的潜在应用。
关键词:人二氢乳清酸脱氢酶;癌症;白血病;黑色素瘤;病毒人二氢乳清酸脱氢酶(Human dihydroorotate dehydrogenase, hDHODH)将二氢乳清酸(L-dihydroorotate,DHO)转化为乳清酸(rotate,0R0),是嘧啶核苷酸从头合成途径第四步反应的关键酶,抑制hDHODH则可抑制嘧啶的从头合成,而细胞的快速增殖需要大量的从头合成嘧啶,因此可通过抑制hDHODH来阻止细胞的快速增殖,从而达到治疗癌症、移植排异、类风湿性关节炎、自身免疫性疾病、抗病毒等的目的。
1癌症hDHODH在DNA形成中的基本作用就是使它成为肿瘤化疗的一个理想靶标。
Brown等人⑴认为阻断TNBC细胞嘧啶合成途径会阻碍他们的DNA修复能力,可使化疗时DNA 损伤效应更敏感。
事实上,他们将TNBC细胞暴露于阿霉素(一种常用的化疗药物)和来氟米特(hDHODH抑制剂)的混合物中,表明来氟米特对异种移植肿瘤有显著的致敏作用。
这些研究提供了关键的临床前证据,由于化疗暴露而诱导的 从头合成嘧啶适应性重编程可被利用,用以提高治疗TNBC 的基因毒性化疗药物的抗癌活性。
P53失活是人类肿瘤发展过程中的常见事件,尽管P53肿瘤抑制因子的许多功能影响线粒体过程,但线粒体生理改变在p53反应调节中的作用仍不清楚。
apoa5基因敲除仓鼠模型的构建方法

apoa5基因敲除仓鼠模型的构建方法
ApoA5基因是人体中一个重要的脂质代谢相关基因,它编码的蛋白质可以调节血浆三酰甘油水平。
因此,研究ApoA5基因的功能和作用对于了解脂质代谢的机制以及相关疾病的发生发展具有重要意义。
为了更好地研究ApoA5基因的功能,科学家们利用基因敲除技术构建了ApoA5基因敲除仓鼠模型。
ApoA5基因敲除仓鼠模型的构建方法主要包括以下几个步骤:
1.设计ApoA5基因敲除的引物
首先,科学家们需要设计一对特异性引物,用于扩增ApoA5基因的外显子序列。
引物的设计需要考虑到引物的长度、GC含量、Tm值等因素,以确保引物的特异性和扩增效率。
2.构建ApoA5基因敲除载体
将引物扩增得到的ApoA5基因外显子序列克隆到敲除载体中,构建ApoA5基因敲除载体。
敲除载体通常包括两个同向的荧光蛋白基因,用于筛选敲除细胞株。
3.转染和筛选
将ApoA5基因敲除载体转染到仓鼠胚胎干细胞中,利用荧光蛋白筛选出ApoA5基因敲除细胞株。
筛选出的细胞株需要进行PCR验证和Southern blotting确认。
4.建立ApoA5基因敲除仓鼠模型
将ApoA5基因敲除细胞株注射到野生型仓鼠的受精卵中,通过胚胎移植技术建立ApoA5基因敲除仓鼠模型。
建立的仓鼠模型需要进行PCR验证和Southern blotting确认。
通过以上步骤,科学家们成功地构建了ApoA5基因敲除仓鼠模型。
这个模型可以用于研究ApoA5基因的功能和作用,以及与脂质代谢相关的疾病的发生发展机制。
同时,这个模型也为开发相关疾病的治疗方法提供了新的思路和途径。
拉莫三嗪改善阿尔茨海默病APPswePS1dE9双转基因模型小鼠认知功能的实验研究

拉莫三嗪改善阿尔茨海默病APPswe/PS1dE9双转基因模型小鼠认知功能的实验研究阿尔茨海默病(Alzheimer’s disease, AD)是以p-淀粉样蛋白(β-amyloid,Aβ)沉积与tau蛋白过度磷酸化形成的神经纤维缠结为特征的神经退行性疾病。
不断增多的Ap和神经纤维缠结进一步引起慢性神经炎症反应,导致突触、神经元缺失,以及进行性的认知功能障碍。
目前认为,靶向针对增多的Ap和tau蛋白过度磷酸化是治疗AD的主要策略。
文献报道AD患者表现出更高的癫痫发生率,并且在大量的AD转基因动物模型脑内均发现存在神经网络异常兴奋性的现象。
这种神经网络活动异常兴奋性以及海马代偿性产生的抑制性机制可能是导致AD以及AD转基因动物模型认知障碍的原因。
因此,靶向针对性抑制脑内神经网络的异常兴奋性,可能有效抑制AD的进行性学习与记忆障碍。
Sanchez等人研究发现,抗癫痫药物左乙拉西坦通过抑制脑内神经网络异常兴奋性显著改善mAPPJ20AD转基因小鼠认知能力的作用。
这些研究成果为抗癫痫药物在AD治疗上的运用提供了一种新的策略。
但并不是所有的抗癫痫药物都显示出这样的疗效。
另一些抗癫痫药物反而会加重AD脑内异常的神经网络活动并且导致认知功能进行下降,如钠离子通道抑制剂苯妥英钠、卡马西平、苯巴比妥、普瑞巴林以及苯二氮卓类抗癫痫药物等。
因此,本实验拟研究抗癫痫药物拉莫三嗪能否抑制AD转基因小鼠脑内神经超兴奋性,能否改善记忆功能的作用,并探讨其可能的分子机制。
拉莫三嗪(lamotrigine, LTG)是一种广谱的抗癫痫药物,用于12岁以上儿童及成人具有简单部分性发作、复杂部分性发作、继发性全身强直-阵挛性发作及原发性全身强直-阵挛性发作等癫痫的治疗。
拉莫三嗪尤其对老年女性癫痫患者显示了独特的显著疗效。
此外,LTG还运用于治疗双向躁狂抑郁障碍、精神分裂症以及偏头痛、神经痛等其它神经系统疾病。
更引人注意的是,拉莫三嗪对成年癫痫患者有改善认知功能的作用。
肽类药物及其抗肿瘤的研究进展

肽类药物及其抗肿瘤的研究进展赵微;白丽【摘要】在抗肿瘤治疗中,抗肿瘤肽类药物具有独特的优势:分子量小、不良反应低、抗肿瘤特异性高及免疫原性低,并且易于合成和改造,对于肿瘤的临床治疗将具有重要价值.【期刊名称】《基础医学与临床》【年(卷),期】2014(034)007【总页数】4页(P998-1001)【关键词】肽类;抗肿瘤;免疫调节【作者】赵微;白丽【作者单位】大理学院云南省昆虫生物医药研发重点实验室,云南大理671000;大理学院基础医学院医学微生物学与免疫学教研室,云南大理671000;大理学院云南省昆虫生物医药研发重点实验室,云南大理671000;大理学院基础医学院医学微生物学与免疫学教研室,云南大理671000【正文语种】中文【中图分类】R730.54肿瘤已成为仅次于心血管疾病的第二大人类致死病因,每年死于癌症的患者约占总死亡人数的1/4,且中国占相当庞大的病例数。
药物治疗是当今治疗肿瘤的主要手段之一,但目前的抗肿瘤药物不良反应较大。
对此,研制高效、低毒的抗肿瘤药物,在医药界显得尤为迫切。
随着肿瘤研究的不断进展,基于免疫和分子生物学而发展的,存在于动物、植物、细菌或真菌的小分子肽类,因稳定、活性高和易于吸收,可以通过提高机体免疫功能抑制肿瘤的生长和转移,增强抗肿瘤作用[1],已成为肿瘤治疗研究的一个新热点[2]。
1.1 肽类的来源肽类来源广泛,在植物[3]、动物[4]、细菌及真菌[5]等生物体内均存在。
肽类存在的形式不同,获取的方法也不相同,获取肽类的途径有3种:1)天然肽类,可用生物提取法获得,具有原料来源广、工艺相对简单、产量大等优点。
据报道,从昆虫体内可以提取出多种天然生物活性肽。
如从毒蝎子中提取的天然抗菌肽,对人类癌细胞有很强的抑制作用[6]。
2)蛋白质降解后产生的肽,可用体内消化法和体外水解法[7]产生。
乳铁蛋白肽 (lactoferricin,Lfcin)是动物乳汁中的乳铁蛋白(lactoferrin,Lf)在动物消化道中经酸性胃蛋白酶降解后产生的含25~28个氨基酸的小肽,它具有杀菌、抗病毒、抗真菌等多种独特的生物学活性。
四环素基因表达系统的研究进展

四环素基因表达系统的研究进展发表时间:2018-07-05T13:22:51.317Z 来源:《医药前沿》2018年6月第18期作者:尚冰清[导读] 基因的表达调控,涉及到多个因素的互相作用,是一个较为复杂的过程。
(徐州医科大学临床医学系院江苏徐州 221006)【摘要】四环素基因调控系统(Tet系统)是迄今为止基因治疗研究中使用最广泛的系统,该系统通过改变培养基、体内四环素或其衍生物(如强力霉素)的浓度,诱导或抑制目标基因的表达。
【关键词】基因调控;四环素基因调控系统;基因治疗【中图分类号】R319 【文献标识码】A 【文章编号】2095-1752(2018)18-0005-02Newstrategiesintetracycline—regulatable system Shang Bingqing.Department of Clinical Medicine, Xuzhou Medical University, Xuzhou 221006, China【Abstract】The tetracycline regulatory system(Tet system)is by far the most frequently used in gene therapy.By adjusting theconcentration of tetracycline or its derivatives(such as doxycycline in medium or in vivo,the expression of target gene will be switched on or off.【Key words】Gene regulation;Tet system;Gene therapy基因的表达调控,涉及到多个因素的互相作用,是一个较为复杂的过程。
在基因治疗中,如果对目的基因的表达时间和表达水平能进行精准调控,就能够有效的降低其副作用的发生率,提高治疗效果,为基因治疗提供重要的理论基础。
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* Correspondence: asea@ 1 Department of Pathology, Scott & White Memorial Hospital and Clinic and the Texas A&M Health Science Center, College of Medicine, Temple, TX 76504, USA Full list of author information is available at the end of the article
Kaur et al. BMC Cancer 2012, 12:120 /1471-2407/12/120
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Background Triple-negative breast cancer (TNBC) is characterized by a lack of receptor expression of estrogen receptor (ER) and progesterone receptor (PgR) and lack of gene expression for human epidermal growth factor receptor 2 (HER2) [1,2]. Chemotherapy remains the only possible therapeutic option in the adjuvant or metastatic setting in the TNBC. Importantly, the prognostic effect of TNBC is independent of poor grade, nodal status, tumor size and treatment [3]. The aggressiveness of TNBC is further indicated by the fact that: (i) the peak risk of recurrence occurs within the first 3 years after initial treatment of the disease with the majority of deaths occurring in the first 5 years [4] and (ii) after diagnosis of metastatic disease, a significantly shorter survival is observed in TNBC [4]. Conversely, the risk for late recurrences (i.e. beyond 5 years of diagnosis) is decreased by 50% compared with HER2-positive disease [5]. More than 90% of TNBC exhibit an invasive ductal histology and high histological grade, present with high mitotic index and carry central necrotic zones and pushing borders as well as a conspicuous lymphocytic infiltrate. TNBC are typically observed in young African-American women, Hispanic women and Caucasian women who carry a mutation in the breast cancer 1 (BRCA1) early onset gene [6]. While some TNBC respond to chemotherapy, subsets of TNBC are chemotherapy-resistant and highly metastatic carrying with it an extremely poor prognosis [6]. The molecular mechanism, which governs the aggressive behavior of this subset of TNBC, is a matter of intense speculation, particularly since TNBC frequently express markers of epithelial mesenchymal transition (EMT). EMT is a normal developmental process in which cells of epithelial origin lose epithelial characteristics and polarity and acquire a mesenchymal phenotype associated with increased migratory behavior [7,8]. Self-renewing cancer cells are reported to be the only cell types within a tumor with an unlimited ability to initiate tumor growth and are therefore known as tumor-initiating cells (TICs), cancer stem cells or tumor-propagating cells [9-11]. The majority of carcinoma cells express a CD44low/CD24high phenotype; however, a small subpopulation of carcinoma cells present within human breast cancers that exhibit a CD44high/ CD24 low antigenic phenotype are highly enriched for TICs [12]. TNBC have been reported to have a higher percentage of CD44 high/CD24low expressing cells than other breast cancer subtypes [13]. The mouse twenty-five kDa heat shock protein (Hsp25/ mouse HspB1) belongs to the family of small HSP and is the murine homologue of human twenty-seven kDa heat shock protein (Hsp27/human HspB1) which operates
Kaur et al. BMC Cancer 2012, 12:120 /1471-2407/12/120
EARCH ARTICLE
Open Access
A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease
Punit Kaur1, Ganachari M Nagaraja1, Hongying Zheng1, Dawit Gizachew2, Moses Galukande3, Sunil Krishnan4 and Alexzander Asea1,5*
Abstract
Background: Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease–recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems. Methods: To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis. Results: We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBCTICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24 + /CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A). Conclusions: Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC. Keywords: Triple-negative breast cancer, Mouse and human HspB1, Hsp25, Hsp27, Hsp72/HspA1A, Heat shock, Cancer stem cells, Tumor-initiating cells