Olmutinib_HM71224_Protein Tyrosine Kinase_EGFR-HER2_CAS号1353550-13-6说明书_AbMole中国

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常春藤皂苷元通过调控巨噬细胞Mincle介导的炎症减轻银屑病小鼠皮肤损伤的作用机制

◇基础研究◇摘要目的：观察常春藤皂苷元（hederagenin ，HDG ）改善银屑病小鼠皮肤损伤和炎症的作用与机制。

方法：通过在C57小鼠背部祛毛并连续涂抹咪喹莫特7d 建立小鼠银屑病动物模型，造模后1h 给予HDG 灌胃治疗。

总计设置正常组、模型组、模型+HDG 低剂量（25mg ·kg -1·d -1）、模型+HDG 高剂量（50mg ·kg -1·d -1）和模型+卤米松阳性对照组（每组8只小鼠）。

给药7d 后，对患处皮肤进行病理检测，以及炎症指标进行ELISA 、实时定量PCR 检测，Mincle 及其下游信号进行免疫组织化学、免疫荧光和Western blot 检测。

结果：与模型组比较，HDG 干预组皮肤病理损伤以及炎性细胞浸润均得到不同程度改善；实时定量PCR 和皮肤组织悬液ELISA 结果证实HDG 干预后小鼠皮肤中炎症因子IL-1β、IL-6和TNF-α的mRNA 及蛋白水平均比模型组降低（P <0.01），说明HDG 具有显著抗炎症作用；免疫组织化学和Western blot 结果表明，与正常组相比，模型组小鼠皮肤中Min-cle 的蛋白表达量显著增加（P <0.01），给予HDG 干预后明显下调（P <0.01）；免疫荧光证实模型组皮肤中Mincle 表达与巨噬细胞标志物F4/80共定位；Western blot 实验发现，HDG 在治疗组中不仅下调了Mincle 的蛋白表达，同时也下调了Mincle 下游信号Syk 和NF-κB 的蛋白磷酸化水平。

结论：HDG 可显著改善银屑病小鼠皮肤损伤和巨噬细胞相关炎症，其潜在分子机制可能与下调Min-cle/Syk/NF-κB 信号途径相关。

关键词常春藤皂苷元；Mincle ；皮肤损伤；炎症；银屑病中图分类号：R965.2文献标志码：A文章编号：1009-2501(2023)12-1339-08doi ：10.12092/j.issn.1009-2501.2023.12.003银屑病是一种慢性丘疹鳞状皮肤病，其主要特点是遗传性和复发性，还可能并发其他疾病，如心血管疾病、糖尿病和关节炎等［1-4］。

五味子水提取液对叔丁基过氧化氢诱导神经元衰老保护作用-赵坚毅

55第19卷第1期 2017 年 1 月辽宁中医药大学学报JOURNAL OF LIAONING UNIVERSITY OF TCMVol. 19 No. 1 Jan .，2017细胞衰老是指正常的细胞在通路的调控下能够不可逆的进入生长停滞期的一种状态[1]。

细胞衰老涉及DNA 的损伤与修复、线粒体的能量代谢以及相关基因的调控等方面[2]。

随着我国人口老龄化进程的加快，细胞衰老问题不仅是一个生物问题，更成为了一个重大的社会问题，所以对于延缓细胞衰老的研究更有现实意义。

五味子为木兰科植物五味子[Schisandra chinensis （Turcz.）Baill]的干燥成熟果实，其果实入药，味酸甘性温，入肺、心、肾经，有敛肺固涩、益气生津及补肾宁心的功效[3]。

现代药理学研究表明，五味子具有保肝[4]、抗缺氧、抗疲劳[5]和抗肿瘤[6]等作用。

高文荣等[7]证实五味子水提取液能够对D-半乳糖所致的衰老神经细胞具有保护作用，但是五味子水提取液对t-BHP 所造成的神经元衰老笔者还未见相关报道。

所以本研究在成功培养小鼠原代皮层神经元基础上，观察五味子水提取液对t-BHP 导致的神经元衰老的作用。

1 材料与方法1.1 动物昆明种小鼠，购自辽宁长生生物技术有限公司，合格证号：SCXK（辽）2010-001，光明∶黑暗=1∶1，室温（22±2）℃，相对湿度55%~65%。

将怀孕的母鼠单独放置在一个鼠笼中，待自然分娩后取新生小鼠用于实验。

1.2 试剂五味子水提取液（由辽宁中医药大学制剂实验室提供），用含有10% FBS（fetal calf serum，胎牛血清，Gibco 公司）和1% P/S（penicillin-strepotomycin，青-链霉素，Thermo 公司）的DMEM（Dulbecco's五味子水提取液对叔丁基过氧化氢诱导神经元衰老保护作用赵坚毅，田立东（辽宁中医药大学附属医院，辽宁沈阳 110032）摘要：目的：探讨五味子水提取液（SWE）对叔丁基过氧化氢诱导的神经元衰老的保护作用并探讨其机制。

维生素D与甲状腺疾病的相关研究进展

维生素D与甲状腺疾病的相关研究进展王君;韩亚玲【摘要】维生素D在人体骨钙代谢平衡中具有重要作用,同时还具有骨骼外作用,它是一种新免疫调节激素,几乎在所有身体组织中均可能产生影响,其中维生素D与甲状腺疾病的关系密切.目前,国内外对Graves病及桥本甲状腺炎的基础及临床研究较为关注,尤其是维生素D缺乏与其患病率有一定的相关性,但其在预防及治疗上的价值仍需进一步研究.同时,虽然维生素D在抗肿瘤治疗中的作用已被认可,但其与甲状腺癌的研究相对较少,未来需要广泛的试验来探索两者之间的关系及作用机制.【期刊名称】《医学综述》【年(卷),期】2018(024)021【总页数】6页(P4245-4249,4255)【关键词】甲状腺疾病;维生素D;25-羟维生素D【作者】王君;韩亚玲【作者单位】保定市第一中心医院内分泌科,河北保定071000;保定市第一中心医院内分泌科,河北保定071000【正文语种】中文【中图分类】R581维生素D属于类固醇化合物，其不仅是人体必需的一种脂溶性维生素，也是一种重要的激素前体。

随着人们对维生素D认识的不断加深，目前发现除维持钙磷平衡和骨骼健康外，维生素D还可以在各种非骨骼疾病中发挥作用，可影响多种细胞的增殖、分化和凋亡等过程，调节免疫系统，是一种新的免疫调节激素。

此外，维生素D可通过调节各种免疫炎性细胞的分化，改变其细胞因子谱，从而影响免疫炎症反应进程。

目前，维生素D在肌肉、心血管、糖尿病、癌症、自身免疫和炎症反应等中的作用逐渐被关注，而其与甲状腺疾病的关系成为研究热点。

在常见的甲状腺疾病中，如Graves病、桥本甲状腺炎(Hashimoto′s thyroiditis, HT)等，在发病过程中常有免疫因素的参与，其生物学作用是通过维生素D受体(vitaminD receptor，VDR)蛋白所介导。

多个研究表明，维生素D缺乏和(或)VDR基因多态性可影响免疫细胞的凋亡，导致甲状腺损伤[1-3]。

Antibody structure, instability, and formulation

MINIREVIEWAntibody Structure,Instability,and FormulationWEI WANG,SATISH SINGH,DAVID L.ZENG,KEVIN KING,SANDEEP NEMAPﬁzer,Inc.,Global Biologics,700Chesterﬁeld Parkway West,Chesterﬁeld,Missouri63017Received14March2006;revised17May2006;accepted4June2006Published online in Wiley InterScience().DOI10.1002/jps.20727 ABSTRACT:The number of therapeutic monoclonal antibody in development hasincreased tremendously over the last several years and this trend continues.At presentthere are more than23approved antibodies on the US market and an estimated200ormore are in development.Although antibodies share certain structural similarities,development of commercially viable antibody pharmaceuticals has not been straightfor-ward because of their unique and somewhat unpredictable solution behavior.This articlereviews the structure and function of antibodies and the mechanisms of physical andchemical instabilities.Various aspects of formulation development have been examinedto identify the critical attributes for the stabilization of antibodies.ß2006Wiley-Liss,Inc.and the American Pharmacists Association J Pharm Sci96:1–26,2007Keywords:biotechnology;stabilization;protein formulation;protein aggregation;freeze drying/lyophilizationINTRODUCTIONProtein therapies are entering a new era with the inﬂux of a signiﬁcant number of antibody pharmaceuticals.Generally,protein drugs are effective at low concentrations with less side effects relative to small molecule drugs,even though,in rare cases,protein-induced antibody formation could be serious.1Therefore,this category of therapeutics is gaining tremendous momentum and widespread recognition both in small and large drugﬁrms.Among protein drug therapies,antibodies play a major role in control-ling many types of diseases such as cancer, infectious diseases,allergy,autoimmune dis-eases,and inﬂammation.Since the approval of theﬁrst monoclonal antibody(MAb)product -OKT-3in1986,more than23MAb drug products have entered the market(Tab.1).The estimated number of antibodies and antibody derivatives constitute20%of biopharmaceutical products currently in development(about200).2The global therapeutic antibody market was predicted to reach$16.7billion in2008.3There are several reasons for the increasing popularity of antibodies for commercial develop-ment.First,their action is speciﬁc,generally leading to fewer side effects.Second,antibodies may be conjugated to another therapeutic entity for efﬁcient delivery of this entity to a target site, thus reducing potential side effects.For instance, Mylotarg is an approved chemotherapy agent composed of calicheamicin conjugated to huma-nized IgG4,which binds speciﬁcally to CD33for the treatment of CD33-positive acute myeloid leukemia.Another example is the conjugation of immunotoxic barnase with the light chain of the anti-human ferritin monoclonal antibody F11as potential targeting agents for cancer immuno-therapy.4Third,antibodies may be conjugated to radioisotopes for speciﬁc diagnostic purposes. Examples include CEA-Scan for detection of color-ectal cancer and ProstaScint for detection of prostate stly,technology advancement has made complete human MAb available,which are lessimmunogenic.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY20071 Correspondence to:Wei Wang(Telephone:(636)-247-2111;Fax:(636)-247-5030;E-mail:wei.2.wang@pﬁ)Journal of Pharmaceutical Sciences,Vol.96,1–26(2007)Pharmacists AssociationT a b l e 1.C o m m e r c i a l M o n o c l o n a l A n t i b o d y P r o d u c t s#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H1A v a s t i n B e v a c i z u m a bH u m a n i z e d I g G 1,149k D a2004G e n e t e c h a n d B i o O n c o l o g y I V i n f u s i o nM e t a s t a t i c c a r c i n o m a o f c o l o n o r r e c t u m ,b i n d s V E G F 100m g a n d 400m g /v i a l (25m g /m L )s o l u t i o n 5.8m g /m L m o n o b a s i c N a P h o s H 2O ;1.2m g /m L d i b a s i c N a P h o s a n h y d r o u s (4m L ,16m L ﬁl l i n v i a l )60m g /m L a -T r e h a l o s e d i h y d r a t e (4m L ,16m L ﬁl l i n v i a l )0.4m g /m L P S 20(4m L ,16m L ﬁl l i n v i a l )6.22B e x x a rT o s i t u m o m a b a n d I -131T o s i t u m a b M u r i n e I g G 2l2003C o r i x a a n d G S KI V I n f u s i o nC D 20p o s i t i v e f o l l i c u l a r n o n H o d g k i n s l y m p h o m aK i t :14m g /m L M A b s o l u t i o n i n 35m g a n d 225m g v i a l s ;1.1m g /m L I 131-M A b s o l u t i o n10m M p h o s p h a t e (M A b v i a l )145m M N a C l ,10%w /v M a l t o s e ;I 131-M A b :5–6%P o v i d o n e ,1–2,9–15m g /m L M a l t o s e ,0.9m g /m L N a C l ,0.9–1.3m g /m L A s c o r b i c a c i d 7.23C a m p a t h A l e m t u z u m a bH u m a n i z e d ,I g G 1k ,150k D a2001I l e x O n c o l o g y ;M i l l e n i u m a n d B e r l e xI V i n f u s i o nB -c e l l c h r o n i c l y m p h o c y t i c l e u k e m i a ,CD 52-a n t i g e n 30m g /3m L s 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a s i c N a P h o s Á7H 2O ;0.42m g /m L M o n o b a s i c N a P h o s ÁH 2O8.48m g /m L N a C l 7.0–7.46H e r c e p t i n (l y o )T r a s t u z u m a bH u m a n i z e d I g G 1k1998G e n e t e c h I V i n f u s i o n M e t a s t a t i c b r e a s t c a n c e r w h o s e t u m o r o v e r e x p r e s s H E R 2p r o t e i n 440m g /v i a l ,21m g /m L a f t e r r e c o n s t i t u t i o n 9.9m g /20m L L -H i s t i d i n e H C l ,6.4m g /20m L L -H i s t i d i n e400m g /20m L a -T r e h a l o s e D i h y d r a t e 1.8m g /20m L P S 2067H u m i r a A d a l i m u m a bH u m a n I g G 1k ,148k D a2002C A T a n d A b b o t t S CR A p a t i e n t s n o t r e s p o n d i n g t o D M A R D s .B l o c k s T N F -a l p h a40m g /0.8m L s o l u t i o n (50m g /m L )0.69m g /0.8m L M o n o b a s i c N a P h o s Á2H 2O ;1.22m g /0.8m L D i b a s i c N a P h o s Á2H 2O ;0.24m g /0.8m L N a C i t r a t e ,1.04m g /0.8m L C i t r i c a c i d ÁH 2O 4.93m g /0.8m L N a C l ;9.6m g /0.8m L M a n n n i t o l 0.8m g /0.8m L P S 805.28L u c e n t i s R a n i b i z u m a bH u m a n i z e d I g G 1k f r a g m e n t2006G e n e n t e c h I n t r a v i t r e a l i n j e c t i o n A g e -r e l a t e d m a c u l a r d e g e n e r a t i o n (w e t )10m g /m L s o l u t i o n10m M H i s t i d i n e H C l10%a -T r e h a l o s e -D i h y d r a t e 0.01%P S 205.52WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jps9M y l o t a r g (l y o )G e m t u z u m a b o z o g a m i c i nH u m a n i z e d I g G 4k c o n j u g a t e d w i t h c a l i c h e a m i c i n2000C e l l t e c h a n d W y e t h I V i n f u s i o nH u m a n i z e d A b l i n k e d t o c a l i c h e a m i c i n f o r t r e a t m e n t o f C D 33p o s i t i v e a c u t e m y e l o i d l e u k e m i a 5m g p r o t e i n -e q u i v a l e n t l y o p h i l i z e d p o w d e r /20-m L v i a l M o n o b a s i c a n d d i b a s i c N a P h o s p h a t e D e x t r a n 40,S u c r o s e ,N a C l 10O n c o S c i n tS 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r v i a l )P h o s p h a t e b u f f e r s a l i n e 5–713R a p t i v a (l y o )E f a l i z u m a bH u m a n i z e d I g G 1k2003X o m a a n d G e n e n t e c h S C C h r o n i c m o d e r a t e t o s e v e r e p l a q u e p s o r i a s i s ,b i n d s t o C D 11a s u b u n i t o f L F A -1150m g M A b /v i a l ;125m g /1.25m L (100m g /m L )a f t e r r e c o n s t i t u t i o n w i t h 1.3m L S W F I 6.8m g /v i a l L -H i s t i d i n e H C l ÁH 2O ;4.3m g /v i a l L -H i s t i d i n e123.2m g /v i a l S u c r o s e 3m g /v i a l P S 206.214R e m i c a d e (l y o )I n ﬂi x i m a bC h i m e r i c h u m a n /m u r i n e M A b a g a i n s t T N F a l p h a (a p p .30%m u r i n e ,70%c o r r e s p o n d s t o h u m a n I g G 1h e a v y c h a i n a n d h u m a n k a p p a l i g h t c h a i n c o n s t a n t r e g i o n s )1998C e n t o c o r I V i n f u s i o nR A a n d C r o h n ’s d i s e a s e (a n t i T N F a l p h a )100m g /20-m L V i a l ,10m g /m L o n r e c o n s t i t u t i o n2.2m g /10m L M o n o b a s i c N a P h o s H 2O ,6.1m g /10m L D i b a s i c N a P h o s Á2H 2O 500m g /10m L S u c r o s e 0.5m g /10m L P S 807.215R e o P r o A b c i x i m a bF a b .C h i m e r i c h u m a n -m u r i n e ,48k D a 1994C e n t o c o r /L i l l y I V i n j e c t i o n a n d i n f u s i o n R e d u c t i o n o f a c u t e b l o o d c l o t r e l a t e d c o m p l i c a t i o n s 2m g /m L s o l u t i o n 0.01M N a P h o s p h a t e 0.15M N a C l 0.001%(0.01m g /m L )P S 807.216R i t u x a n R i t u x i m a bC h i m e r i c m o u s e /h u m a n I g G 1k w i t h m u r i n e l i g h t a n d h e a v y c h a i n v a r i a b l e r e g i o n (F a b d o m a i n ),145kD a1997I D E C a n d G e n e n t e c h I V i n f u s i o nN o n H o d g k i n ’s l y m p h o m a .(a n t i C D 20-a n t i g e n )10m g /m L s o l u t i o n7.35m g /m L N a C i t r a t e Á2H 2O9m g /m L N a C l 0.7m g /m L P S 806.5(C o n t i n u e d )ANTIBODY FORMULATION3DOI 10.1002/jpsJOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 200717S i m u l e c t (l y o )B a s i l i x i m a bC h i m a r i c I g G 1k ,144kD a1998N o v a r t i s I V i n j e c t i o n a n d i n f u s i o nP r e v e n t i o n o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n ,I L -2r e c e p t o r a n t a g o n i s t10m g a n d 20m g /v i a l ,4m g /m L o n r e c o n s t i t u t i o n 3.61m g ,7.21m g M o n o b a s i c K P h o s ;0.50m g ,0.99m g N a 2H P O 40.8m g ,1.61m g N a C l ;10m g ,20m g S u c r o s e ;40m g ,80m g M a n n i t o l ;20m g 40m g G l y c i n e 18S y n a g i s (l y o )P a l i v i z u m a bH u m a n i z e d I g G 1k ,C D R o f m u r i n e M A b 1129,148k D a 1998M e d I m m u n e I M i n j e c t i o nP r e v e n t r e p l i c a t i o n o f t h e R e s p i r a t o r y s y n c y t i a l v i r u s (R S V )50m g a n d 100m g /v i a l ,100m g /m L o n r e c o n s t i t u t i o n47m M H i s t i d i n e ,3.0m M G l y c i n e 5.6%M a n n i t o l19T y s a b r i N a t a l i z u m a bH u m a i n z e d I g G 4k2004B i o g e n I D E C I V I n f u s i o nM S r e l a p s e 300m g /15m L s o l u t i o n 17.0m g M o n o b a s i c N a P h o s ÁH 2O ,7.24m g d i B a s i c N a P h o s Á7H 2O f o r 15m L 123m g /15m L N a C l3.0m g /15m L P S 806.120V e r l u m a N o f e t u m o m a b M u r i n e F a b 1996B o e h r i n g e r I n g e l h e i m a n d D u P o n t M e r c k I V i n j e c t i o n I m a g i n g a g e n t f o r l u n g c a n c e r10m g /m L s o l u t i o nP h o s p h a t e b u f f e r s a l i n e?21X o l a i r (l y o )O m a l i z u m a bH u m a n i z e d I g G 1k ,149k D aG e n e n t e c h w N o v a r t i s a n d T a n o xS CA s t h m a ,i n h i b i t s b i n d i n g o f I g E t o I g E r e c e p t o r F C e R I202.5m g /v i a l ,D e l i v e r 150m g /1.2m L o n r e c o n s t i t u t i o n w i t h 1.4m L S W F I 2.8m g L H i s t i d i n e H C l ÁH 2O ;1.8m g L H i s t i d i n e145.5m g S u c r o s e 0.5m g P S 2022Z e n a p a x D a c l i z u m a bH u m a n i z e d I g G 1,144k D a1997R o c h e I V i n f u s i o nP r o p h y l a x i s o f a c u t e o r g a n r e j e c t i o n i n p a t i e n t s r e c e i v i n g r e n a l t r a n s p l a n t s .I n h i b i t s I L -2b i n d i n g t o t h e T a c s u b u n i t o f I L -2r e c e p t o r c o m p l e x 25m g /5m L M A b S o l u t i o n3.6m g /m L M o n o b a s i c N a P h o s ÁH 2O ;11m g /m L D i b a s i c N a P h o s Á7H 2O4.6m g /m L N a C l 0.2m g /m L P S 806.923Z e v a l i nI b r i t u m o m a b -T i u x e t a nM u r i n e I g G 1k -t h i o u r e a c o v a l e n t l i n k a g e t o T i u x e t a nI D E C I V i n f u s i o nC D 20a n t i g e n .(K i t w i t h Y t t e r i u m -90i n d u c e s c e l l u l a r d a m a g e b y b e t a e m i s s i o n )3.2m g /2m L s o l u t i o n 09%N a C l 7.1T a b l e 1.(C o n t i n u e d )#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H4WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jpsDevelopment of commercially viable antibody pharmaceuticals has,however,not been straight-forward.This is because the behavior of antibodies seems to vary,even though they have similar structures.In attempting to address some of the challenges in developing antibody therapeutics, Harris et al.5reviewed the commercial-scale formulation and characterization of therapeutic recombinant antibodies.In a different review, antibody production and puriﬁcation have been discussed.2Nevertheless,the overall instability and stabilization of antibody drug candidates have not been carefully examined in the litera-ture.This article,not meant to be exhaustive, intends to review the structure and functions of antibodies,discuss their instabilities,and sum-marize the methods for stabilizing/formulating antibodies.ANTIBODY STRUCTUREAntibodies(immunoglobulins)are roughly Y-shaped molecules or combination of such molecules(Fig.1). Their structures are divided into two regions—the variable(V)region(top of the Y)deﬁning antigen-binding properties and the constant(C)region (stem of the Y),interacting with effector cells and molecules.Immunoglobulins can be divided into ﬁve different classesÀIgA,IgD,IgE,IgM,and IgG based on their C regions,respectively desig-nated as a,d,e,m,and g(ﬁve main heavy-chain classes).6Most IgGs are monomers,but IgA and IgM are respectively,dimmers and pentamers linked by J chains.IgGs are the most abundant,widely used for therapeutic purposes,and their structures will be discussed as antibody examples in detail.Primary StructureThe structure of IgGs have been thoroughly reviewed.6The features of the primary structure of antibodies include heavy and light chains, glycosylation,disulﬁde bond,and heterogeneity. Heavy and Light ChainsIgGs contain two identical heavy(H,50kDa)and two identical light(L,25kDa)chains(Fig.1). Therefore,the total molecular weight is approxi-mately150kDa.There are several disulﬁde bonds linking the two heavy chains,linking the heavy and light chains,and residing inside the chains (also see next section).IgGs are further divided into several subclasses—IgG1,IgG2,IgG3,and IgG4(in order of relative abundance in human plasma),with different heavy chains,named g1, g2,g3,and g4,respectively.The structural differences among these subtypes are the number and location of interchain disulﬁde bonds and the length of the hinge region.The light chains consist of two types—lambda(l)and kappa(k). In mice,the average of k to l ratio is20:1,whereas it is2:1in humans.6The variable(V)regions of both chains cover approximately theﬁrst 110amino acids,forming the antigen-binding (Fab)regions,whereas the remaining sequences are constant(C)regions,forming Fc(fragment crystallizable)regions for effector recognition and binding.6The N-terminal sequences of both the heavy and light chains vary greatly between different antibodies.It was suggested that the conserved sequences in human IgG1antibodies Figure1.Linear(upper panel)and steric(lower panel)structures of immunoglobulins(IgG).ANTIBODY FORMULATION5DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007are approximately95%and the remaining5% is variable and creates their antigen-binding speciﬁcity.5The V regions are further divided into three hypervariable sequences(HV1,HV2,and HV3)on both H and L chains.In the light chains,these are roughly from residues28to35,from49to59,and from92to103,respectively.6Other regions are the framework regions(FR1,FR2,FR3,and FR4).The HV regions are also called the complementarity determining regions(CDR1,CDR2,and CDR3). While the framework regions form the b-sheets, the HV sequences form three loops at the outer edge of the b barrel(also see Section2.2).Disulﬁde BondsMost IgGs have four interchain disulﬁde bonds—two connecting the two H chains at the hinge region and the other two connecting the two L chains to the H chains.6Exceptions do exist.Two disulﬁde bonds were found in IgG1and IgG4 linking the two heavy chain in the hinge region but four in IgG2.7In IgG1MAb,HC is linked to the LC between theﬁfth Cys(C217)of HC and C213on the LC.In IgG2and IgG4MAbs,it is the third Cys of HC(C123)linking to the LC.7A disulﬁde bond between HC C128and LC C214 was found for mouse catalytic monoclonal anti-bodies(IgG2a).8IgGs have four intrachain disulﬁde bonds, residing in each domain of the H and L chains, stabilizing these domains.The intrachain disul-ﬁde bonds in V H and V L are required in functional antigen binding.9Native IgG MAbs should not have any free sulfhydryl groups.7However, detailed examination of the free sulfhydryl groups in recombinant MAbs(one IgG1,two IgG2,and one IgG4)suggests presence of a small portion of free sulfhydryl group(approximately0.02mol per mole of IgG2or IgG4MAb and0.03for IgG1.7In rare cases,a free cysteine is found.A nondisulﬁde-bonded Cys at residue105was found on the heavy chain of a mouse monoclonal antibody,OKT3 (IgG2a).10OligosaccharidesThere is one oligosaccharide chain in IgGs.6This N-linked biantennary sugar chain resides mostly on the conserved Asn297,which is buried between the C H2domains.5,11For example,the oligosaccharide resides on Asn-297of the C H2 domain of chimeric IgG1and IgG3molecules12but on Asn299in a monoclonal antibody,OKT3 (IgG2a).10The oligosaccharide,often microheter-ogeneous,is typically fucosylated in antibodies produced in CHO or myeloma cell lines5and may differ in other cell lines.2,11There are many factors that dictate the nature of the glycan microheterogenity on IgGs.These include cell line,the bioreactor conditions and the nature of the downstream processing.An additional oligo-saccharide can be found in rare cases.A human IgG produced by a human-human-mouse hetero-hybridoma contains an additional oligosaccharide on Asn75in the variable region of its heavy chain.13In addition,O-linked carbohydrates could also exist in this antibody.Proper glycosylation is critical for correct functioning of antibodies.11It was demonstrated that removal of the oligosaccharide in IgGs(IgG1 and IgG3)made them ineffective in binding to C1q, in binding to the human Fc g RI and activating C; and generally more sensitive to most proteases than their corresponding wild-type IgGs(one exception).12This is because the binding site on IgG for C1q,theﬁrst component of the complement cascade,is localized in the C H2domains.11 Furthermore,the glycosylation can affect the antibody conformation.12Oligosaccharides in other regions can also play a critical role.Removal of an oligosaccharide in a Fv region of the CBGA1antibody resulted in a decreased antigen-binding activity in several ELISA systems.13In addition,this oligosaccharide might play critical role in reducing the antigenicity of the protein.14The sugar composition of the oligosaccharide is also critical in antibody functions.It has been shown that a low fucose(Fuc)content in the complex-type oligosaccharide in a humanized chimeric IgG1is responsible for a50-fold higher antibody-dependent cellular cytotoxicity(ADCC) compared with a high Fuc counterpart.15 HeterogeneityPuriﬁed antibodies are heterogeneous in struc-ture.This is true for all monoclonal antibodies (MAbs)due to differences in glycosylation pat-terns,instability during production,and terminal processing.5For example,ﬁve charged isoforms were found in recombinant humanized monoclo-nal antibody HER2as found by capillary iso-electric focusing(cIEF)and sodium dodecyl sulfate–capillary gel electrophoresis(SDS–CGE).16Six separate bands were focused under6WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jpsIEF for two mouse monoclonal antibodies IgG2a (k)and IgG1(k).17A mature monoclonal antibody, OKT3(IgG2a),contain cyclized N-terminus (pyroglutamic acid,À17D)in both H and L chains, processed C-terminus(no Lys,À128D)of the H chains,and a small amount of deamidated form.10 Similar observation was also reported for a huma-nized IgG1(k).18In rare cases,gene cross-over may lead to formation of abnormal heavy chains.For example,a puriﬁed monoclonal anti-IgE antibody contains a small amount of a variant H chain, which had16fewer amino acid residues than the normal H chain(position is between Arg108of the L chain and Ala124of the H chain).19 Secondary and Higher-Order StructureThe basic secondary and higher-order structural features of IgGs have been reviewed.6Only a small portion of the three-dimensional structures of IgGs has been solved.20The antibody’s secon-day structure is formed as the polypeptide chains form anti-parallel b-sheets.The major type of secondary structure in IgGs is these b-sheets and its content is roughly70%as measured by FTIR.21The light chain consists of two and the heavy chain contains four domains,each about 110amino acid long.6,20All these domains have similar folded structures—b barrel,also called immunoglobulin fold,which is stabilized by a disulﬁde bond and hydrophobic interaction(pri-mary).These individual domains($12kDa in size)interact with one another(V H and V L;C H1 and C L;and between two C H3domains except the carbohydrate-containing C H2domain)and fold into three equal-sized spherical shape linked by a ﬂexible hinge region.These three spheres form a Y shape(mostly)and/or a T shape.22The less globular shape of IgGs is maintained both by disulﬁde bonds and by strong noncovalent interactions between the two heavy chains and between each of the heavy-chain/light-chain pairs.23Through noncovalent interactions,a less stable domain becomes more stable,and thus,the whole molecule can be stabilized.24A detailed study indicates that the interaction between two CH3domains are dominated by six contact residues,ﬁve of these residues(T366,L368, F405,Y407,and K409)forming a patch at the center of the interface.25These noncovalent interactions are spatially oriented such that variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)induces noncovalent multimerization.26The six hypervariable regions in CDR(L1,L2, L3,H1,H2,and H3)form loops of a few predictable main-chain conformations(or canonical forms), except H3loop,which has too many variations in conformation to be predicted accurately.27,28 There is a slight difference in the loop composition and shape between the two types of light chains.20 However,no functional difference was found in antibodies having l or k chain.6Basic Functions of AntibodiesThe basic functions of antibodies have been reviewed.6There are two functional areas in IgGs—the V and C regions.The V regions of the two heavy and light chains offer two identical antigen-binding sites.The binding of the two sites (bivalent)can be independent of each other and does not seem to depend on the C region.29The exact antigen-binding sites are the CDR regions with participation of the frame work regions.30 Binding of antigens seems through the induced-ﬁt mechanism.31,32The induced-ﬁt mechanism allows multispeciﬁcity and polyreactivity.It has been suggested that about5–10residues usually contribute signiﬁcantly to the binding energy.32 The C regions of antibodies have three main effector functions(1)being recognized by receptors on immune effector cells,initiating antibody-dependent cell cytotoxicities(ADCC),(2)binding to complement,helping to recruit activated pha-gocytes,and(3)being transported to a variety of places,such as tears and milk.6In addition,C domains also modulate in vivo stability.23,29,33The function of Fc is affected by the structure of Fab. Variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)affected Fc-associated func-tions such as serum half-life and binding to protein G and Fc g RI.26The hinge region providesﬂexibility in bivalent antigen binding and activation of Fc effector functions.26Two chimeric IgG3antibodies lacking a genetic hinge but with Cys residues in CH2 regions was found to be deﬁcient in their inter-molecular assembly,and both IgG3D HþCys and IgG3D Hþ2Cys lost greatly their ability to bind Fc g RI and failed to bind C1q and activate the complement cascade.34Alternative Forms of AntibodiesIn addition to species-speciﬁc antibodies,other antibody forms are generated to meet various needs.In the early development of antibody therapies,antibodies were made from murineANTIBODY FORMULATION7DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007sources.However,these antibodies easily elicit formation of human anti-mouse antibody (HAMA).Therefore,humanized chimeric antibo-dies were generated.Chimeric monoclonal anti-bodies(60–70%human)are made of mouse variable regions and human constant regions.2 Such antibodies can still induce formation of human anti-chimeric antibody(HACA).Highly humanized antibodies,CDR-grafted antibodies, are made by replacing only the human CDR with mouse CDR regions(90–95%human).2These antibodies are almost the same in immunogeni-city potential as completely human antibodies, which may illicit formation of human anti-human antibody(HAHA).Other alternative forms of antibodies have also been generated and these different forms have been reviewed.35Treatment with papain would cleave the N-terminal side of the disulﬁde bonds and generate two identical Fab fragments and one Fc fragment.Fab0s are50kDa(V HþC H1)/ (V LþC L)heterodimers linked by a single disul-ﬁde bond.Treatment with pepsin cleaves the C-terminal side of the disulﬁde bonds and pro-duces a F(ab)02fragment.The remaining H chains were cut into several small fragments.6Cleavage by papain occurs at the C-terminal side of His-H22836or His-H227.37Reduction of F(ab0)2will produce two Fab0.23Fv fragments are noncovalent heterodimers of V H and V L.Stabilization of the fragment by a hydrophilicﬂexible peptide linker generates single-chain Fv(scFvs).2Fragments without constant domains can also be made into domain antibodies (dAbs).These scFvs are25–30kDa variable domain (V HþV L)dimers joined by polypeptide linkers of at least12residues.Shorter linkers(5–10residues)do not allow pairing of the variable domains but allow association with another scFv form a bivalent dimer (diabody)(about60kDa,or trimer:triabody about 90kDa).38Two diabodies can be further linked together to generate bispeciﬁc tandem diabody (tandab).39Disulﬁde-free scFv molecules are rela-tively stable and useful for intracellular applica-tions of antibodies—‘‘intrabodies.’’38The smallest of the antibody fragments is the minimal recognition unit(MRU)that can be derived from the peptide sequences of a single CDR.2ANTIBODY INSTABILITYAntibodies,like other proteins,are prone to a variety of physical and chemical degradation path-ways,although antibodies,on the average,seem to be more stable than other proteins.Antibody instabilities can be observed in liquid,frozen,and lyophilized states.The glycosylation state of an antibody can signiﬁcantly affect its degradation rate.40In many cases,multiple degradation path-ways can occur at the same time and the degrada-tion mechanism may change depending on the stress conditions.41These degradation pathways are divided into two major categories—physical and chemical instabilities.This section will explore the possible degradation pathways of antibodies and their inﬂuencing factors.Physical InstabilityAntibodies can show physical instability via two major pathways—denaturation and aggregation. DenaturationAntibodies can denature under a variety of conditions.These conditions include temperature change,shear,and various processing steps. Compared with other proteins,antibodies seem to be more resistant to thermal stress.They may not melt completely until temperature is raised above708C,21,42,43while most other mesophilic proteins seem to melt below708C.44Shear may cause antibody denaturation.For example,the antigen-binding activity of a recombinant scFv antibody fragment was reduced with aﬁrst-order rate constant of0.83/h in a buffer solution at a shear of approximately20,000/s.45Lyophilization can denature a protein to var-ious extents.An anti-idiotypic antibody(MMA 383)in a formulation containing mannitol,sac-charose,NaCl,and phosphate was found to loose its in vivo immunogenic properties(only10–20% of normal response rate)upon lyophilization.46 Since the protein showed no evidence of degrada-tion after lyophilization,no change in secondary structure by CD(29%b-sheet,14%a-helix,and 57%‘‘other’’),the loss of activity was attributed to the conformational change.Indeed,tryptophan ﬂuorescence properties were different between the lyophilized and unlyophilized antibodies.46 AggregationAntibody aggregation is a more common manifes-tation of physical instability.The concentration-dependent antibody aggregation was considered the greatest challenge to developing protein formulations at higher concentrations.47This is8WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jps。

《临床肝胆病杂志》推荐使用的规范医学名词术语

临床肝胆病杂志第40卷第3期2024年3月J Clin Hepatol， Vol.40 No.3， Mar.2024[3]XIA SL, LIU ZM, CAI JR, et al. Liver fibrosis therapy based on biomi⁃metic nanoparticles which deplete activated hepatic stellate cells[J]. J Control Release, 2023, 355: 54-67. DOI: 10.1016/j.jconrel.2023.01.052.[4]LIU YW, DONG YT, WU XJ, et al. The assessment of mesenchymalstem cells therapy in acute on chronic liver failure and chronic liver disease: A systematic review and meta-analysis of randomized con⁃trolled clinical trials[J]. Stem Cell Res Ther, 2022, 13(1): 204. DOI:10.1186/s13287-022-02882-4.[5]ZHANG ZL, SHANG J, YANG QY, et al. Exosomes derived from hu⁃man adipose mesenchymal stem cells ameliorate hepatic fibrosis by inhibiting PI3K/Akt/mTOR pathway and remodeling choline me⁃tabolism[J]. J Nanobiotechnology, 2023, 21(1): 29. DOI: 10.1186/ s12951-023-01788-4.[6]ZHAO T, SU ZP, LI YC, et al. Chitinase-3 like-protein-1 function andits role in diseases[J]. Signal Transduct Target Ther, 2020, 5(1): 201. DOI: 10.1038/s41392-020-00303-7.[7]YANG H, ZHAO LL, HAN P, et al. Value of serum chitinase-3-likeprotein 1 in predicting the risk of decompensation events in patients with liver cirrhosis[J]. J Clin Hepatol, 2023, 39(7): 1578-1585. DOI:10.3969/j.issn.1001-5256.2023.07.011.杨航, 赵黎莉, 韩萍, 等. 血清壳多糖酶3样蛋白1(CHI3L1)对肝硬化患者发生失代偿事件风险的预测价值[J]. 临床肝胆病杂志, 2023, 39(7): 1578-1585. DOI: 10.3969/j.issn.1001-5256.2023.07.011.[8]MA L, WEI J, ZENG Y, et al. Mesenchymal stem cell-originated exo⁃somal circDIDO1 suppresses hepatic stellate cell activation by miR-141-3p/PTEN/AKT pathway in human liver fibrosis[J]. Drug Deliv, 2022, 29(1): 440-453. DOI: 10.1080/10717544.2022.2030428. [9]NISHIMURA N, DE BATTISTA D, MCGIVERN DR, et al. Chitinase 3-like 1 is a profibrogenic factor overexpressed in the aging liver and in patients with liver cirrhosis[J]. Proc Natl Acad Sci U S A, 2021, 118(17): e2019633118. DOI: 10.1073/pnas.2019633118.[10]WANG CG, LI SZ, SHI JM, et al. Research progress in differentia⁃tion, identification, and purification methods of human pluripotent stem cells to mesenchymal-like cells in vitro[J]. J Jilin Univ Med Ed, 2023, 49(6): 1655-1661. DOI: 10.13481/j.1671-587X.20230634.王成刚, 李生振, 史嘉敏, 等. 体外人多能干细胞向间充质样细胞分化、鉴定和纯化方法的研究进展[J]. 吉林大学学报(医学版), 2023, 49(6): 1655-1661. DOI: 10.13481/j.1671-587X.20230634.[11]LI TT, WANG ZR, YAO WQ, et al. Stem cell therapies for chronicliver diseases: Progress and challenges[J]. Stem Cells Transl Med, 2022, 11(9): 900-911. DOI: 10.1093/stcltm/szac053.[12]YANG X, LI Q, LIU WT, et al. Mesenchymal stromal cells in hepaticfibrosis/cirrhosis: From pathogenesis to treatment[J]. Cell Mol Im⁃munol, 2023, 20(6): 583-599. DOI: 10.1038/s41423-023-00983-5. [13]ZHAO SX, LIU Y, PU ZH. Bone marrow mesenchymal stem cell-derived exosomes attenuate D-GaIN/LPS-induced hepatocyte apop⁃tosis by activating autophagy in vitro[J]. Drug Des Devel Ther, 2019, 13: 2887-2897. DOI: 10.2147/DDDT.S220190.[14]LEE CG, HARTL D, LEE GR, et al. Role of breast regression protein39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue re⁃sponses and apoptosis[J]. J Exp Med, 2009, 206(5): 1149-1166.DOI: 10.1084/jem.20081271.[15]HIGASHIYAMA M, TOMITA K, SUGIHARA N, et al. Chitinase 3-like 1deficiency ameliorates liver fibrosis by promoting hepatic macro⁃phage apoptosis[J]. Hepatol Res, 2019, 49(11): 1316-1328. DOI:10.1111/hepr.13396.收稿日期：2023-06-09；录用日期：2023-08-17本文编辑：邢翔宇引证本文：LIU PJ, YAO LC, HU X, et al. Effect of human umbilical cord mesenchymal stem cells in treatment of mice with liver fibrosis and its mechanism[J]. J Clin Hepatol, 2024, 40(3): 527-532.刘平箕, 姚黎超, 胡雪, 等. 人脐带间充质干细胞(hUC-MSC)对肝纤维化小鼠模型的治疗作用及其机制分析[J]. 临床肝胆病杂志, 2024, 40(3): 527-532.读者·作者·编者《临床肝胆病杂志》推荐使用的规范医学名词术语有关名词术语应规范统一，以全国自然科学名词审定委员会公布的各学科名词为准。

G蛋白耦联胆汁酸受体激动剂INT777通过激活AMPK信号通路抑制施万细胞成髓鞘过程

南通大学学报(医学版)Journal of Nantong University (Medical Sciences) 2021 : 41 (1)6・D0I:10.16424/32-1807/r.2021.01.002G 蛋白耦联胆汁酸受体激动剂INT777通过激活AMPK 信号通路抑制施万细胞成髓鞘过程*关晋东：丁杰，刘晓宇，孙诚**** ［基金项目］国家自然科学基金青年基金资助项目(81701222)** ［作者简介］关晋东，男，汉族，生于1995年10月，山西省晋城市人，硕士在读，研究方向:外周神经发育及损伤修复机制的研究。

*** ［通信作者］孙诚，电话**************,E-mail: ********************.cn(南通大学教育部/江苏省神经再生重点实验室/神经再生协同创新中心，南通226001)［摘要］目的：研究G 蛋白耦联胆汁酸受体(G-protein-coupled bile acid receptor 1, GPBAR1,同时也被称为TGR5) 特异性激动剂6琢-乙基-23(S)-甲基胆酸［6 alpha-ethyl-23(S)-methylcholic acid, INT777［对原代施万细胞髓鞘形成的影响并初步探讨其可能的作用机制遥方法:用5 滋mol/L 的INT777处理二丁酰环腺苷酸(dibutyryl cyclic adenoslne phosphate, dbcAMP )诱导分化原代施万细胞成髓鞘模型，用免疫印迹(Western Blot )方法检测髓磷脂蛋白表达量的变化遥同时，提取施万细胞总核糖核酸后用定量聚合酶链式反应试验检测INT777对髓鞘形成过程相关分子基因表达的影响遥另外，用Western Blot 方法检测INT777对单磷酸腺苷活化蛋白激酶/核糖体蛋白S6激酶(adenosine 5'-monophosphate-activatedprotein kinase/ribosomal S6 kinase, AMPK/S6K )信号途径的影响遥结果:在dbcAMP 诱导分化原代施万细胞成髓鞘过程中，5 滋mol/L INT777的处理抑制了髓鞘早期生长因子20,八聚体结合转录因子6,髓磷脂蛋白的表达，且5滋mol/L INT777 处理激活了 AMPK 的活性，抑制了雷帕霉素作用靶点信号通路遥结论：INT777抑制dbcAMP 诱导的施万细胞成髓鞘过程，这种抑制作用可能是通过激活施万细胞AMPK 活性、抑制S6K 活性实现的遥［关键词］施万细胞曰髓鞘曰6琢-乙基-23(S)-甲基胆酸曰单磷酸腺苷活化蛋白激酶［中图分类号］R338.1 ［文献标志码］A ［文章编号］1674-7887(2021)01-0006-05G-protein-coupled bile acid receptor agonists INT777 inhibits myelination of Schwann cells byactivating AMPK signaling pathway*GUAN Jindong **, DING Jie, LIU Xiaoyu, SUN Cheng ***('Key Laboratory of Neuroregeneration of Jiangsu and Ministry ofEducation, Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226001)［Abstract ］ Objective: To investigate the effects of G-protein-coupled bile acid receptor 1(GPBAR1, also known as TGR5)specific agonist 6 alpha-ethyl-23(S)-methylcholic acid(INT777) on myelination in primary Schwann cells and the underlying mechanisms. Methods: Primary Schwann cells were treated with 5 滋mol/L INT777 and dibutiryl cyclic adenoslne phosphate (dbcAMP), and the changes of myelin protein zero were detected by Western Blot. Meanwhile, the total RNA was extractedfrom Schwann cells and quantitative real time polymerase chain reaction was employed for detecting myelin gene expression. In addition, the effect of INT777 on the adenosine 5' -monophosphate -activated protein kinase/ribosomal protein S6 kinase (AMPK/S6K) signaling pathway was examined by Western Blot. Results : Treatment with 5 滋mol/L INT777 inhibited theexpression of myelin early growth response -2, octamer -binding transcription factor 6, and myelin protein zero during dbcAMP-induced myelination of differentiated Schwann cells, and treatment with 5 滋mol/L INT777 activated AMPK activity and inhibited mTOR signaling pathway. Conclusion: INT777 attenuates dbcAMP-induced myelination of Schwann cells, whichmay be achieved by activating AMPK and inhibiting S6K activity.［Key words ］ Schwann cell; myelination; 6 alpha-ethyl -23(S)-methylcholic acid; adenosine 5'-monophosphate-activated protein kinase外周神经系统(peripheral nervous system, PNS) 在机体内分布广泛且起到介导靶器官与中枢神经系统信号传递的重要作用。

枸橼酸他莫昔芬与鲑鱼精DNA和牛血清白蛋白的相互作用分析

枸橼酸他莫昔芬与鲑鱼精DNA和牛血清白蛋白的相互作用分析摘要：通过实验得知，蛋白质与药物的结合非常的容易；枸橼酸他莫昔芬与鲑鱼精DNA和牛血清白蛋白的缔合方式只有1种，即嵌插缔合。

最后由热力学常数计算得出之间的相互作用为疏水作用。

关键词：枸橼酸他莫昔芬鲑鱼精DNA 牛血清白蛋白枸橼酸他莫昔芬属三苯乙烯非甾体抗雌激素类抗肿瘤药物。

在晚期乳腺癌、绝经时期，它的应用对男性前列腺癌和卵巢癌的治疗作用非常的显著。

一直以来，小分子与DNA分子相互作用的研究备受关注，而分子和DNA的相互作用有剪切作用、共价结合、非共价结合3种模式；其中沟槽作用、嵌插缔合、静电作用为非共价结合，而嵌插缔合作用的研究最为广泛。

人和牛的血清白蛋白结构比较的相似，为此牛的血清白蛋白可用于对小分子和蛋白质相互作用的研究之中，在药理学上，这种相互作用的意义非常的重要，通过相互作用可以对药物毒性、生物活性进行研究。

1 实验（一）实验设备实验所用的光度计分别为北京普析通用仪器有限责任公司生产的TU-1810紫外-可见分光光度计和美国PerkinElmer公司生产的Lambda 35荧光分光光度计。

pH值为7.2的Tris-HCL缓冲溶液；1×10-3mol/L的TC（C32H37NO8）溶液，在使用之前要用Tris缓冲溶液进行稀释；根据260nm下测定的ε260=6600L/(mol·cm)的浓度确定的鲑鱼精DNA，且在4℃的冰箱中进行保存；1×10-5mol/L的BSA。

另外，实验所用的试剂为分析纯，二次去离子水作为用水进行实验。

（二）方法使用Tris-HCl缓冲溶液对TC样品进行稀释，使其浓度为2.2×10-6mol/L，并在样品中滴加ss-DNA溶液后对其吸光度进行测试；使用Tris-HCl缓冲溶液对TC样品进行稀释，使其浓度为1.76×10-6mol/L，进行ss-DNA溶液的逐次滴加后，对荧光强度进行测试。

仑伐替尼在肝细胞癌中的耐药机制和对策研究进展

收稿日期:２０２３Ｇ０８Ｇ２１基金项目:国家自然科学基金项目(８２０６０４３５)作者简介:徐永康(１９９６ ),男,博士研究生,主要从事肝癌的临床和基础研究.通信作者:吴建兵,主任医师,E Ｇm a i l :h h g w jb ＠１６３．c o m .仑伐替尼在肝细胞癌中的耐药机制和对策研究进展徐永康,吴建兵(南昌大学第二附属医院肿瘤科,南昌３３０００６)摘要:仑伐替尼是晚期肝癌有效的一线治疗药物.然而,许多患者在仑伐替尼治疗后出现疾病进展,耐药问题在治疗实践中普遍存在,临床急需逆转耐药策略.众多研究表明表观遗传学㊁细胞转运过程㊁肿瘤微环境㊁肿瘤干性以及细胞自噬㊁铁死亡等与仑伐替尼的耐药机制密切相关.文章对肝癌仑伐替尼耐药的机制研究进展进行归纳,并初步探讨耐药后临床治疗策略,旨在为改善仑伐替尼耐药患者预后提供理论基础.关键词:肝细胞癌;仑伐替尼;耐药性;临床策略中图分类号:R ７３５．７㊀㊀㊀文献标志码:A㊀㊀㊀文章编号:２０９５Ｇ４７２７(２０２４)０２－００８１－０７D O I :１０．１３７６４/j．c n k i ．n c d m．２０２４．０２．０１４R e s i s t a n c e o fL e n v a t i n i b a n d I t sU s e i n t h eT r e a t m e n to fH e pa t o c e l l u l a rC a r c i n o m a :aR e v i e w X UY o n g Ｇk a n g ,W UJ i a n Ｇb i n g(D e p a r t m e n t o f O n c o l o g y ,t h eS e c o n dA f f i l i a t e d H o s p i t a l o f N a n c h a n gU n i v e r s i t y ,N a n c h a n g ３３０００６,C h i n a )A B S T R A C T :L e n v a t i n i b i s a n e f f e c t i v e f i r s t Ｇl i n e t h e r a p e u t i c a g e n t f o r a d v a n c e d h e pa t o c e l l u l a r c a r Ｇc i n o m a ．H o w e v e r ,m a n y p a t i e n t s e x p e r i e n c e d i s e a s e p r o g r e s s i o n a f t e r l e n v a t i n ib t r e a t m e n t ,a n d t h e p r o b l e mo f d r u g r e s i s t a nc e i s p r e v a l e n t i n t h e r a p e u t i c p r a c t i c e ,u r g e n t l y r e q u i r i n g s t r a t e gi e s t o r e Ｇv e r s ed r u g r e s i s t a n c e ．N u m e r o u ss t u d i e sh a v es h o w nt h a te p i g e n e t i c s ,c e l l t r a n s po r t p r o c e s s e s ,t u m o rm i c r o e n v i r o n m e n t ,t u m o r d r y n e s s ,a sw e l l a s a u t o p h a g y a n d f e r r o p t o s i s ,a r e c l o s e l y re l a t e d t o t h e r e s i s t a n c e m e c h a n i s m of l e n v a t i n i b ．T h i sa r t i c l es u mm a r i z e s t h er e s e a r c h p r o gr e s so nt h e m e c h a n i s mo f l e n v a t i n i b r e s i s t a n c e i nh e p a t o c e l l u l a r c a r c i n o m a ,a n d p r e l i m i n a r i l y e x p l o r e s c l i n i c a l t r e a t m e n t s t r a t e g i e s t o p r o v i d e a t h e o r e t i c a l b a s i s f o r i m p r o v i n g t h e p r o g n o s i s o f l e n v a t i n i b Ｇr e s i s t Ｇa n t p a t i e n t s ．K E Y W O R D S :h e p a t o c e l l u l a r c a r c i n o m a ;l e n v a t i n i b ;d r u g r e s i s t a n c e ;c l i n i c a l s t r a t e g y㊀㊀据国际癌症研究机构的估算,到２０４０年,全球新发肝癌病例预计将达到１４０万例,相较于２０２０年将增长５５％;而全球肝癌导致的死亡人数也预计将达到１３０万人,较２０２０年增加５６．４％[１].肝细胞癌(H C C )是最常见的肝癌类型,约占肝癌病例的９０％[２].当前,全球和中国的肝癌防治形势十分严峻,因此提高肝癌的诊断和治疗水平对于我国的肝癌患者尤为重要.根据R E F L E C T 研究显示,与索拉非尼相比,仑伐替尼在总生存期(O S )㊁客观缓解率(O R R )(２４．１％比９．２％)和无进展生存期(P F S )方面表现出非劣效(P F S７．４个月比３．７个月)[３].在晚期肝癌的系统治疗中,我国广泛使用仑伐替尼,并且取得了显著的疗效.然而,随着治疗时间的延长,１８南昌大学学报(医学版)２０２４年第６４卷第２期㊀J o u r n a l o fN a n c h a n g U n i v e r s i t y(M e d i c a l S c i e n c e s )２０２４,V o l ．６４N o ．２肿瘤耐药问题不可避免,导致患者疾病进展.因此,探索仑伐替尼耐药的机制以及新的耐药途径显得尤为迫切.另外,目前临床上对于仑伐替尼耐药患者的治疗方案尚无统一标准.本文对仑伐替尼治疗H C C耐药机制以及耐药后的临床研究进展进行综述,以期为临床提高参考.１㊀表观遗传与仑伐替尼耐药㊀㊀表观遗传是一种与核苷酸序列变化无关但能够改变生物表型的重要机制.已有大量证据显示,异常的表观遗传调控可能导致肿瘤耐药的发生[４].因此,对于仑伐替尼耐药与表观遗传机制之间的关系进行深入阐述,将为未来逆转耐药策略提供理论基础.１．１㊀R N A修饰R N A修饰是表观遗传学调控中的重要组成部分,包括甲基化修饰㊁羟甲基化修饰㊁乙酰化修饰和磷酸化修饰等常见类型,近年来发现这些修饰在仑伐替尼耐药形成过程中扮演着重要角色.在仑伐替尼耐药细胞中,m６A修饰相关蛋白M E T T L３表达显著上调,进一步机制研究[５]发现,M E T T L３通过m６A修饰调节E G F R的m R N A翻译,针对M E TＧT L３的特异性抑制剂S T M２４５７可以提高细胞对仑伐替尼的敏感性.此外,有研究[６]还发现在耐药细胞中m７G甲基转移酶复合物的关键成分M E T T L１和WD R４上调,通过介导t R N A m７G修饰和调控T R I M２８表达等途径促进了对仑伐替尼的耐药性[７].另外,N A T１０的上调可以通过调节H S P９０A A１m R N A a c４C的修饰水平维持H S P９０A A１的稳定性,促进了内质网应激H C C的转移和仑伐替尼耐药作用[８].此外,Y R D C也在调节K R A S蛋白质翻译方面发挥作用,其低t６A修饰水平的t R N A降低了K R A S的翻译,从而介导肝癌细胞对仑伐替尼的耐药[９].当前,R N A修饰蛋白成为了治疗肿瘤耐药的新靶标,针对M E T T L３的小分子抑制剂已经应用于临床研究,为提升仑伐替尼治疗反应性提供了新的选择.１．２㊀非编码R N A非编码R N A是基因表达调控的关键因素之一.微小R N A s(m i R N A s)㊁长链非编码R N A s(l nＧc R N A s)和环状R N A s(c i r c R N A s)通过影响转录㊁转录后修饰和翻译等生物学过程,对癌症的发生发展和肿瘤耐药发挥着双重作用[１０].１．２．１㊀m i c r o R N A在仑伐替尼耐药的人肝癌细胞株H u h７和S MM CＧ７７２１中,CＧM e t呈现过度表达和活化的现象.有研究[１１]表明,m i RＧ１２８Ｇ３p通过抑制cＧM e t表达,调节介导凋亡途径的A k t和调节细胞周期进程的E R K参与了仑伐替尼的耐药机制.此外,在仑伐替尼耐药的肝癌细胞中,A K R１C１和m i c r o R N A ４６４表达水平显著上调.数据库分析提示A K R１C１和m i c r o R N A４６４可能存在靶向关系,而m i c r o RＧN A４６４被认为是早期诊断仑伐替尼耐药性的一个潜在生物标志物[１２].１．２．２㊀l n c R N A在仑伐替尼耐药的H C C细胞中,L n c R N A MT１J P(MT１J P)呈现上调的情况,导致了MT１J P 和抗凋亡蛋白B C L２L２的过度表达,从而降低了H C C细胞对仑伐替尼的敏感性[１３].MT１J P通过吸附m i RＧ２４Ｇ３p来靶向B C L２L２,进而促进了仑伐替尼的耐药性.此外,有研究[１４]报道了l n c X I S T通过激活H C C细胞中的E Z H２ＧN O D２ＧE R K轴来促进仑伐替尼的耐药性.S O N G等[１５]观察到在仑伐替尼耐药的细胞和组织中P I N K１的高表达,这有助于维持线粒体的结构和功能,并促进抗氧化应激反应,进一步的机制研究发现F G D５ＧA S１/m i RＧ５５９０Ｇ３p/P I N K１轴可以促进肝癌细胞对仑伐替尼的耐药性.机制研究表明L I N C０１６０７/m i RＧ８９２b/P６２促进了线粒体自噬,降低了R O S水平,从而导致肿瘤的耐药性.最后,敲除L I N C０１６０７联合仑伐替尼能够有效地克服类器官模型中仑伐替尼的耐药性[１６].１．２．３㊀c i r c R N A通过对２８例接受仑伐替尼治疗后出现耐药的患者进行外周血检测,发现c i r c M E D２７水平显著上升.相关研究[１７]表明,c i r c M E D２７作为m i RＧ６５５Ｇ３p的竞争内源性R N A发挥着重要作用,其上调促使U S P２８的表达增加,在肝癌的进展和仑伐替尼耐药过程中发挥关键作用.此外,L I U等[１８]首次发现c i r c K C N N２通过m i RＧ５２０cＧ３p/M B D２轴抑制了H C C的复发.潜在的机制可能在于c i r c K C N N２和仑伐替尼都能够抑制F G F R４的表达,从而加强对仑伐替尼的抗肿瘤效果.H A O等[１９]研究发现C i rＧc P A K１通过H i p p o信号通路促进了Y A P的核转运,从而促进了H C C的进展.同时,该研究发现在仑伐替尼耐药的L M３ＧL R和H e pＧ３BＧL R H C C细胞中,C i r c P A K１的表达上调.外泌体中高表达的C i r c P A K１从耐药细胞转运到敏感细胞,诱导细胞对仑伐替尼的耐药性.非编码R N A作为治疗靶点在改善肿瘤耐药方面显示出极具竞争力和前景.然而,在药物的稳定性和传递效率方面仍然存在挑战.２８南昌大学学报(医学版)２０２４年４月,第６４卷第２期２㊀信号通路与仑伐替尼耐药㊀㊀作为一种多靶点酪氨酸激酶抑制剂,仑伐替尼主要作用于血管内皮生长因子(V E G F R)１Ｇ３㊁成纤维细胞生长因子受体(F G F R)１Ｇ４等靶点,从而发挥抗肿瘤的作用.近期的研究逐渐揭示,仑伐替尼治疗导致V E G F R和F G F R的抑制,进而引发E G F R/ S T A T３㊁R A S/R A F/M E K/E R K㊁P I３K/A K T等相关信号通路的反馈激活.这些信号通路的激活被认为是引发肿瘤对靶向治疗产生耐药的关键机制.因此,通过针对H C C内部相关信号通路的交叉对话进行靶向阻断,成为解决耐药问题的一种有效策略.２．１㊀E G F R介导的信号通路J I N等[２０]通过C R I S P RＧC a s９基因筛选,确定了E G F R作为仑伐替尼的合成致死靶点.研究机制显示,仑伐替尼治疗抑制F G F R,进而导致E G F RＧP A K２ＧE R K５信号轴的反馈激活.另一方面,HU 等[２１]的研究表明,A B C B１在E G F R激活下以脂质筏依赖的方式被激活,从而显著增强了仑伐替尼的细胞排出作用.这种激活通过激活E G F R并刺激E G F RＧS T A T３ＧA B C B１轴,导致对仑伐替尼产生耐药性.考虑到E G F R通路在肝癌靶向治疗中的关键作用,L I M等[２２]利用c t D N A分析了肝癌患者在治疗前和治疗进展时E G F R途径的遗传变化.他们发现,在仑伐替尼治疗进展期间,患者显示出E GＧF R的拷贝数增加(４．８拷贝Ｇ７拷贝)和E R B B２的激活突变(S３１０Y).这些E G F R/E R B B２的遗传改变可能是导致仑伐替尼耐药性的遗传机制之一.２．２㊀R A S/M E K/E R K信号通路耐药细胞中V E G F R２的表达以及其下游R A S/M E K/E R K信号轴显著上调,E T SＧ１被确认为介导V E G F R２相关的仑伐替尼耐药的原因.此外,槐定碱被发现能够降低E T SＧ１的表达,从而抑制耐药H C C细胞中V E G F R２及其下游R A S/ M E K/E R K轴的表达[２３].L U等[２４]发现N F１和D U S P９在H C C中是仑伐替尼耐药的关键驱动因素,研究者通过R N A i敲除和C R I S P R/C a s９敲除模型进一步阐明了N F１激活P I３K/A K T和MA P K/E R K信号通路的机制.另外,D U S P９激活MA P K/E R K信号通路,导致F O X O３失活降解,最终诱导耐药发生.在仑伐替尼耐药细胞中,发现３种血管生成细胞因子(V E G F㊁P D G FＧA A㊁A n g)显著上调,加速了肿瘤血管生成,增加了获得性耐药的发生,同时观察到了MA P K/M E K/E R K信号通路的激活和E MT标记物的上调[２５].２．３㊀其他信号通路HO U等[２６]发现,I T G B８通过H S P９０介导的A K T的稳定以及A K T信号的增强来调节仑伐替尼的耐药性.通过使用A K T抑制剂MKＧ２２０６或H S P９０抑制剂１７ＧA A G,可以使耐药细胞重新对仑伐替尼治疗产生敏感性.此外,另一项研究[２７]指出,T HO C２可能通过W n t/βＧc a t e n i n通路参与调控肝癌对仑伐替尼的耐药性.肿瘤细胞异常信号通路的激活在获得性耐药中是常见的机制,针对这些关键靶点进行有效干预是防治耐药的有效策略.例如,已证实E G F R抑制剂㊁cＧM E T抑制剂以及M E K抑制剂在一定程度上可以逆转耐药现象,有望成为临床上的新对策.３㊀肿瘤微环境与仑伐替尼耐药㊀㊀肿瘤微环境构成了一个极为复杂的生态系统,由各种细胞㊁非细胞成分和分泌产物组成,它们之间的相互作用形成了复杂的保护和修复机制,从而导致肿瘤产生耐药性.因此,深入探索新的针对微环境的靶向策略和药物,以改善耐药性并抑制肿瘤的恶性增殖显得尤为重要.作为一种抗血管生成药物,长期使用仑伐替尼可能会导致肿瘤细胞缺氧,部分细胞因此产生耐药性.在缺氧条件下,H I FＧ１α诱导P L C/P R F/５细胞中纤维连接蛋白的生成并削弱了仑伐替尼的作用,进而导致耐药性的产生.进一步的研究[２８]表明,联合抑制纤维连接蛋白和MA P K通路可能是一种有效的治疗策略.肿瘤相关成纤维细胞(C A F)长期处于激活状态,形成致密的纤维间质包绕瘤块,通过分泌可溶性因子和直接细胞间接触等途径,对耐药性的形成起着潜在作用.研究人员发现,C A F和H C C细胞的共培养显著降低了H C C细胞对索拉非尼/仑伐替尼的体内外反应性.C A F分泌的S P P１通过P K Cα信号通路激活R A F/MA P K和P I３K/A K T/m T O R 通路,并通过E MT增强了肝癌患者对酪氨酸激酶抑制剂的抵抗.因此,治疗前血浆S P P１水平可能成为预测治疗反应的潜在生物标志物[２９].４㊀细胞自噬与仑伐替尼耐药㊀㊀自噬是一种细胞死亡编程,与肿瘤的恶性演变和耐药性密切相关,过去的研究[３０Ｇ３１]已经表明在肝癌索拉非尼耐药中自噬发挥着关键作用,近期对仑伐替尼耐药机制的探索中也发现了类似的现象. P A N等[３２]证实L A P T M５的上调会导致细胞自噬水平上升,从而降低H C C对仑伐替尼的敏感性,而３８徐永康等:仑伐替尼在肝细胞癌中的耐药机制和对策研究进展通过L A P T M５的沉默或者使用H C Q来阻断内在的自噬潮,可以与仑伐替尼协同作用来抑制H C C 的生长.此外,HO A I R M１在仑伐替尼耐药细胞系中被发现是一个独立的耐药因子,与仑伐替尼治疗H C C的疗效显著相关.敲低HO T A I R M１可以增加H u h７ＧR和H e p G２ＧR细胞中m i RＧ３４a的水平,并抑制B e c l i nＧ１的表达.因此,HO T A I R M１可能通过下调m i RＧ３４a和上调B e c l i nＧ１来诱导自噬的激活,从而导致H C C对仑伐替尼的耐药性[３３].５㊀铁死亡与仑伐替尼耐药㊀㊀铁死亡是一种铁依赖性的细胞死亡方式,诸多证据表明此过程与肿瘤治疗耐药相关联,调节铁死亡过程可能有效改善耐药性.I S E D A等[３４]分析了仑伐替尼对H C C细胞的细胞毒性,并证实了仑伐替尼通过抑制F G F R４,抑制x C T表达并诱导脂质R O S积累的方式诱导H C C细胞发生铁死亡.此外,活化的N r f２抑制了仑伐替尼诱导的铁死亡,因此抑制N r f２的活化有望提高H C C细胞对仑伐替尼的敏感性,从而改善肝癌的耐药性.另一项研究[３５]发现,K E A P１是驱动索拉非尼耐药的关键基因,K E A P１失活导致了K E A P１/N r f２途径的失活,通过上调N r f２下游基因和降低R O S水平,导致人类H C C细胞对索拉非尼的耐药性增加.同时,该研究还发现K E A P１的破坏抑制了仑伐替尼诱导的细胞活力下降,同时降低了对药物反应产生R O S的能力,这表明K E A P１也可能是仑伐替尼的易感基因之一.６㊀其他机制６．１㊀肿瘤细胞干性肿瘤干细胞(C S C)是一种功能细胞状态,具有自我复制和多谱系分化能力,在肿瘤药物抵抗中发挥关键作用,其耐药机制主要涉及药物转运子高表达㊁强D N A修复能力和募集保护性微环境等方面[３６].WA N G等[３７]研究发现F Z D１０在肝C S C中高度表达,从机制上看,M E T T L３介导F Z D１０m RＧN A的m６A修饰,而m６A读取器Y T H D F２随后与F Z D１０m R N A上的m６A位点结合,保持该m R N A 的稳定性.F Z D１０通过WN T/βＧc a t e n i n和H i p p o 途径增强肝脏C S C的特性,并且F Z D１０/βＧc a t e n i n/ cＧJ u n/M E K/E R K轴促进肝癌细胞对仑伐替尼产生耐药性.采用腺相关病毒靶向F Z D１０或βＧc a t e n i n 抑制剂治疗仑伐替尼耐药H C C可恢复其抗肿瘤反应.另一项研究[３８]表明,T M４S F１通过上调MY H９来调节N O T C H通路,进而促进肝癌中干细胞干性和仑伐替尼耐药性,且C D７３通过上调S O X９的表达和增强其蛋白稳定性,在维持C S C性状方面发挥关键作用.C D７３的过度表达使H C C 细胞对仑伐替尼产生显著的耐药性,同时也在维持肝癌索拉非尼和卡博替尼的耐药性中发挥作用.因此,将C D７３作为靶点可能是根除C S C和逆转肝癌患者对T K I耐药性的一种有希望的策略[３９].６．２㊀糖酵解H C C在缺氧和营养缺乏的环境中,通过适应性机制 W a r b u r g效应 ,优先依靠糖酵解产生能量,相关的转运蛋白㊁关键限速酶和代谢产物能够通过多种机制促使肿瘤进展和耐药.因此,探索糖酵解调控的耐药机制对于癌症治疗具有重要意义.A CＧY P１在糖酵解中扮演直接调节的角色,并通过A CＧY P１/H S P９０/MY C/L D H A轴驱动了仑伐替尼耐药性和H C C的进展,靶向A C Y P１可以与仑伐替尼协同作用,更有效地治疗H C C[４０].另外,果糖Ｇ２,６Ｇ二磷酸酶３(P F K F B３)是糖酵解的有效刺激剂, P F K F B３的表达上调可以导致H C C细胞对仑伐替尼的耐药.联合使用P F K F B３抑制剂可以在仑伐替尼耐药后抑制P F K F B３及MM P s的表达,从而逆转H C C细胞对仑伐替尼的耐药性[４１].７㊀逆转耐药的机制策略和临床对策７．１㊀机制策略７．１．１㊀E G F R抑制剂J I N等[２０]在体外和体内实验中,利用E G F R抑制剂吉非替尼和仑伐替尼的联合作用于表达E G F R 的肝癌细胞系㊁异种肝癌细胞系㊁免疫活性小鼠模型以及病人来源H C C异种移植肿瘤,均观察到显著的抗增殖效果.进一步的临床试验(N C T０４６４２５４７)结果显示,对于１２例经过仑伐替尼治疗后肿瘤仍然进展的E G F R高表达肝癌患者,采用仑伐替尼和吉非替尼联合治疗后,４例部分缓解,４例病情稳定,显示出良好的应用前景.此外, S U N等[４２]的研究表明,E G F R抑制剂吉非替尼与仑伐替尼联合应用能够延缓仑伐替尼耐药细胞的增殖并诱导其凋亡,其机制可能是通过抑制E G F R介导的M E K/E R K和P I３K/A K T通路激活.在体内实验中,仑伐替尼与依拉昔达或吉非替尼联合给药抑制了肿瘤生长和血管生成.HU等[２１]发现另一种E G F R抑制剂厄洛替尼可以抑制A B C B１,从而减少仑伐替尼的胞吐,在体外和体内实验中均表现出对H C C的抑制效果.７．１．２㊀M E K抑制剂L U等[２４]发现一种小分子M E K途径抑制剂曲４８南昌大学学报(医学版)２０２４年４月,第６４卷第２期美替尼可逆转H C C细胞中N F１和D U S P９丢失引起的耐药性,即使在小鼠中敲除N F１,曲美替尼仍然能有效阻止H C C的增殖.此外,HU A N G等[４３]发现M E K抑制剂S e l u m e t i n i b能够消除D U S P４缺乏引起的仑伐替尼耐药性,表明M E K磷酸化和D U S P４抑制依赖性E R K激活是其耐药性产生的关键.７．１．３㊀其他有研究[４４]表明,双硫仑联合铜离子能够增加仑伐替尼对仑伐替尼耐药肝癌细胞H u h７的敏感性,其机制可能与抑制P I３K/A k t通路及促进c a s p a s eＧ９蛋白表达有关.H AMA Y A等[４５]发现化疗也是改善耐药的有效方式,顺铂能够抑制仑伐替尼耐药细胞的增殖,并诱导G２/M细胞周期阻滞.此外,顺铂通过A T M/A T RＧC h k１/C h k２信号通路触发D N A损伤反应.另外,Z H A O等[２３]发现槐定碱可以进一步提高仑伐替尼治疗仑伐替尼耐药H C C的敏感性.７．２㊀仑伐替尼耐药临床对策仑伐替尼已被证明是晚期H C C患者的一线治疗药物,在临床实践中得到了广泛应用.然而,一旦患者出现对仑伐替尼的耐药,目前尚缺乏标准有效的二线治疗方案.当患者出现仑伐替尼耐药时,通常是各临床中心根据自身实际经验选择治疗方式.７．２．１㊀分子靶向治疗抗血管生成药物作为治疗肝癌的重要药物,在仑伐替尼耐药后仍然是临床实践中的有效手段.根据当前的指南推荐,索拉非尼㊁瑞戈非尼㊁雷莫芦单抗等均是仑伐替尼治疗耐药患者的备用方案.T OMO N A R I等[４６]首次报道了１３名在仑伐替尼进展后接受索拉非尼治疗的患者.根据m R EＧC I S T标准,O R R和D C R分别为１５．３％(２/１３)和６９．２％(９/１３).而根据R E C I S T标准,O R R和D C R分别为０％(０/１３)和６９．２％(９/１３),中位P F S 为４．１个月.一项系统评价[４７]分析了４０５４名患者的２０项研究,发现进展后生存期(P P S)与总生存期(O S)之间存在更强的相关性(r＝０．８６９,P＜０．００１).该评价同时探讨了P P S在仑伐替尼耐药患者中的作用,２５例接受二线索拉非尼治疗的患者的O R R和D C R分别为１２％(３/２５)和５２％(１３/２５),P F S为５．７个月(９５％C I:０．８~１０．６个月).另外,一项来自韩国的研究[４８]提示,续贯索拉非尼是一种潜在的治疗选择.该研究发现,索拉非尼治疗的患者的中位O S明显长于接受纳武利尤单抗治疗的患者的中位O S(８．７v s３．０个月;P＝０．０４６).索拉非尼治疗与较低的死亡率相关(H R＝０．１９４;９５％C I:０．０５３~０．７０８;P＝０．０１３).另外,７例H C C患者在仑伐替尼失败后接受雷莫芦单抗作为二线或三线治疗,D C R为２８．６％(２/７例),中位P F S为４１d,具有抑制先前接受仑伐替尼治疗的患者H C C进展以及在治疗期间维持肝功能的潜力[４９].由于肝癌患者发病机制的复杂性,导致药物疗效存在差异性,因此,目前临床对于药物耐药评价以及换药指征把控存在一定差异.一些临床工作者认为,仑伐替尼的高缓解率和低毒性仍会使患者受益.一项通过倾向性匹配来自１１家不同医疗机构的临床数据的研究[５０]发现,相较于其他治疗(包括最佳支持治疗㊁索拉非尼㊁瑞戈非尼㊁雷莫芦单抗),继续仑伐替尼治疗的患者可以获得更好的O S(１０．８/１９．６v s ５．８/１１．２个月,P＜０．００１).７．２．２㊀免疫联合靶向治疗多项免疫联合靶向治疗的优异疗效已经深刻改变了肝癌的系统治疗格局,这些相互联合的治疗方案能够增强治疗效果,其在晚期二线治疗中的应用也在逐渐增加.Z O U等[５１]进行了一项回顾性调查,评估了标准剂量的仑伐替尼与P DＧ１抑制剂的联合治疗对于仑伐替尼治疗进展患者的有效性和安全性,他们发现O R R和D C R分别为２３．９％(１１/４６)和７１．７％(３３/４６),而中位P F S和O S分别为６．９个月和１４．５个月.最常见的治疗相关不良事件包括厌食症(４３．５％)㊁甲状腺功能减退症(４３．５％)和高血压(３６．９％),而３/４级不良事件的发生率为３４．８％(１６/４６).另一项研究[５２]则比较了在仑伐替尼治疗失败患者中,P DＧ１联合仑伐替尼和瑞戈非尼治疗的效果,结果显示联合组的中位P F S和D C R 较单药组有所提高(P F S８．７v s４．２个月,P＝０．０１８;D C R８２．７％v s５３．３％,P＝０．０１).然而,联合组的O S并未显示出显著的获益(１５．３v sN E个月,P＝０．５),且O R R也未显著高于瑞戈非尼组(２７．６％v s１３．３％,P＝０．４９).此外,瑞戈非尼组和联合组的３/４级治疗相关不良反应发生率分别为２６．７％和１０．３％,最常见的不良反应包括丙氨酸氨基转移酶升高㊁疼痛和总胆红素升高.然而,来自广州医科大学附属第二医院朱教授团队[５３]和笔者团队的研究[５４]显示,瑞戈非尼联合P DＧ１抑制剂相较于瑞戈非尼单药治疗在索拉非尼及仑伐替尼一线治疗失败后具有更高的O R R㊁更长的P F S和更好的O S.不过,纳入一线仑伐替尼治疗失败的患者比例较低可能是导致这些研究结果不一致的原因之一.目前,文献报道索拉非尼或免疫联合靶向药５８徐永康等:仑伐替尼在肝细胞癌中的耐药机制和对策研究进展物可能存在一定的获益,但缺乏充分的临床数据支持.仑伐替尼耐药后的治疗方案仍处于探索阶段,但包括C h i C T R２２０００６２８５４㊁C h i C T R２００００３６６６４㊁N C T０５７１８８８２㊁N C T０４６４２５４７等临床试验正在进行中.期待未来会有更多的大型临床研究集中于仑伐替尼耐药患者,以及探索免疫治疗联合仑伐替尼耐药后的治疗方案选择.参考文献:[１]㊀R UM G A Y H,A R N O L D M,F E R L A YJ,e t a l．G l o b a l b u r d e n o f p r i m a r y l i v e rc a n c e r i n２０２０a n d p r e d i c t i o n s t o２０４０[J]．JH e p a t o l,２０２２,７７(６):１５９８Ｇ１６０６．[２]V O G E LA,M E Y E R T,S A P I S O C H I N G,e t a l．H e p a t o c e l l u l a rc a r c i n o m a[J]．L a n c e t,２０２２,４００(１０３６０):１３４５Ｇ１３６２．[３]K U D O M,F I N N R S,Q I N S K,e ta l．L e n v a t i n i bv e r s u ss o rＧa f e n ib i n f i r s tＧl i n e t r e a t m e n t o f p a t i e n t sw i t hu n r e s ec t a b l e h e pＧa t o c e l l u l a r c a r c i n o m a:a r a n d o m i s e d p h a s e３n o nＧi n f e r i o r i t y t r iＧa l[J]．L a n c e t,２０１８,３９１(１０１２６):１１６３Ｇ１１７３．[４]WA N G N,MA T,Y U B．T a r g e t i n g e p i g e n e t i c r e g u l a t o r s t ooＧv e r c o m e d r u g r e s i s t a n c e i n c a n c e r s[J]．S i g n a l T r a n s d u c t T a r g e t T h e r,２０２３,８(１):６９．[５]WA N G L N,Y A N G Q X,Z HO U Q 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s i o ns u r v i v a l o no u t c o m e s o fL e n v a t i n i b t r e a t m e n t f o ru n r e s e c t a b l eh e p a t o c e l l u l a rc a r c i n o m a:a s y s t e m a t i cr e v i e wa n d r e t r o s p e c t i v ec o h o r ts t u d y[J]．A n t i c a n c e rR e s,２０２２,４２(１２):６００７Ｇ６０１８．[４８]K I M Y,L E EJ S,L E EH W,e t a l．S o r a f e n i b v e r s u s n i v o l u m a ba f t e rL e n v a t i n i bt r e a t m e n t f a i l u r e i n p a t i e n t sw i t ha d v a n c e dh e p a t o c e l l u l a rc a r c i n o m a[J]．E u rJ G a s t r o e n t e r o l H e p a t o l,２０２３,３５(２):１９１Ｇ１９７．[４９]K A S U Y A K,K AWAMU R A Y,K O B A Y A S H IM,e t a l．E f f iＧc a c y a nd s a fe t y of r a m u c i r u m a b i n p a t i e n t sw i t hu n r e s e c t a b l eh e p a t o c e l l u l a rc a r c i n o m a w i t h p r o g r e s s i o n a f t e rt r e a t m e n tw i t hL e n v a t i n i b[J]．I n t e r n M e d,２０２１,６０(３):３４５Ｇ３５１．[５０]H I R A O K A A,K UMA D A T,T A D A T,e ta l．W h a tc a nb ed o ne t os o l v e t h eu n m e t c l i n i c a l n e e dof h e p a t o c e l l u l a r c a r c iＧn o m a p a t i e n t s f o l l o w i n g L e n v a t i n i b f a i l u r e?[J]．L i v e rC a n cＧe r,２０２１,１０(２):１１５Ｇ１２５．[５１]Z O UJX,HU A N GPX,G E N L,e t a l．A n t iＧP DＧ１a n t i b o d i e s p l u s l e n v a t i n i bi n p a t i e n t s w i t h u n r e s e c t a b l eh e p a t o c e l l u l a rc a r c i n o m aw h o p r o g r e s s e do nL e n v a t i n i b:ar e t r o s p e c t i v ec oＧh o r t s t u d y o fr e a lＧw o r l d p a t i e n t s[J]．J G a s t r o i n t e s t O n c o l,２０２２,１３(４):１８９８Ｇ１９０６．[５２]G U A NRG,M E I J,L I SH,e t a l．C o m p a r a t i v e e f f i c a c y o f P DＧ１i n h i b i t o r s p l u sL e n v a t i n i ba n dr e g o r a f e n i ba f t e rL e n v a t i n i bf a i l u r ef o ra d v a n c e d h e p a t o c e l l u l a rc a r c i n o m a:ar e a lＧw o r l ds t u d y[J]．H e p a t o l I n t,２０２３,１７(３):７６５Ｇ７６９．[５３]HU A N G JJ,G U O Y J,HU A N G W S,e ta l．R e g o r a f e n i bc o m b i n ed w i t h P DＧ１b l o c k a d ei mm u n o t he r a p y v e r s u sr e g oＧr a f e n i ba ss e c o n dＧl i n et r e a t m e n t f o ra d v a n c e dh e p a t o c e l l u l a rc a r c i n o m a:am u l t i c e n t e r r e t r o s p e c t i v e s t ud y[J]．JHe p a t o c e l lC a r c i n o m a,２０２２,９:１５７Ｇ１７０．[５４]B A R B I E R I I,K O U Z A R I D E S T．R o l eo fR N A m o d i f i c a t i o n si n c a n c e r[J]．N a tR e vC a n c e r,２０２０,２０(６):３０３Ｇ３２２．(责任编辑:李松旻)７８徐永康等:仑伐替尼在肝细胞癌中的耐药机制和对策研究进展。

《2024年穿龙薯蓣皂苷对胶原诱导性关节炎小鼠CD4~+T细胞亚群平衡的影响及其调控机制研究》范文

《穿龙薯蓣皂苷对胶原诱导性关节炎小鼠CD4~+T细胞亚群平衡的影响及其调控机制研究》篇一摘要：本研究探讨了穿龙薯蓣皂苷对胶原诱导性关节炎（CIA）小鼠模型中CD4~+T细胞亚群平衡的影响及其可能的调控机制。

结果表明，穿龙薯蓣皂苷可以有效地调整CD4~+T细胞亚群的失衡，改善小鼠关节炎的病理过程，且该效应可能与其调节细胞信号转导、促进抗炎和抗增殖的作用相关。

一、引言关节炎是一种常见的慢性自身免疫性疾病，其发病机制复杂，涉及免疫系统紊乱。

穿龙薯蓣皂苷作为一种天然植物提取物，具有多种生物活性，包括抗炎、抗氧化和免疫调节等作用。

本研究旨在探讨穿龙薯蓣皂苷对胶原诱导性关节炎（CIA）小鼠模型中CD4~+T细胞亚群平衡的影响及其调控机制。

二、材料与方法1. 实验材料：穿龙薯蓣皂苷、CIA小鼠模型、相关试剂与仪器等。

2. 实验方法：（1）建立CIA小鼠模型；（2）分组及给药：将小鼠随机分为正常对照组、模型组、穿龙薯蓣皂苷治疗组；（3）检测各组小鼠CD4~+T细胞亚群平衡；（4）分析穿龙薯蓣皂苷对CIA小鼠的病理过程的影响；（5）研究穿龙薯蓣皂苷的调控机制。

三、实验结果1. 穿龙薯蓣皂苷对CIA小鼠CD4~+T细胞亚群平衡的影响：穿龙薯蓣皂苷治疗组小鼠的CD4~+T细胞亚群平衡得到显著改善，与模型组相比，治疗组中Th1/Th2比例趋于正常，Th17细胞数量减少。

2. 穿龙薯蓣皂苷对CIA小鼠病理过程的影响：穿龙薯蓣皂苷治疗组小鼠的关节炎症状得到明显缓解，关节肿胀程度减轻，关节滑膜炎症反应减弱。

3. 穿龙薯蓣皂苷的调控机制研究：（1）穿龙薯蓣皂苷可能通过调节细胞信号转导途径，如NF-κB、MAPK等，从而影响CD4~+T细胞的活化与分化；（2）穿龙薯蓣皂苷可能通过促进抗炎和抗增殖的作用，抑制关节炎症反应和关节损伤；（3）穿龙薯蓣皂苷还可能通过调节免疫相关基因的表达，如IL-17、IFN-γ等，进一步影响CD4~+T细胞的亚群平衡。

大分享：肺癌三代EGFR-TKI靶向药及试用机会

大分享：肺癌三代EGFR-TKI靶向药及试用机会肺癌病人如果检测到EGFR突变，适合使用EGFR-TKI 类靶向药治疗。

EGFR（表皮生长因子受体，也叫ErbB1或HER1）的异常激活，是驱动肺癌生长增殖的重要致癌分子机制，抑制EGFR是控制肺癌的重要策略。

EGFR-TKI（表皮生长因子受体-酪氨酸激酶抑制剂）就是这样一类可以靶向抑制EGFR的药物总称。

尽管这类药物的作用靶点都可针对EGFR，但每个药物分子与靶点分子的相互作用机制不尽相同。

正是基于这类药物与靶点作用特征的不断改进和迭代，EGFR-TKI目前可分为第一、二、三代产品。

第一代EGFR-TKI的作用特点为可逆性、非选择性抑制EGFR。

也就是说，既可抑制突变的EGFR，也可抑制未突变（野生型）的EGFR，并且这种抑制作用是可逆的，有许多影响因素可以逆转解除抑制。

一代EGFR-TKI的代表药物包括：吉非替尼、厄洛替尼、埃克替尼等。

第二代EGFR-TKI的作用特点为不可逆性、非选择性、ErbB 受体家族阻断剂（泛-HER抑制剂）。

也就是说，不仅抑制EGFR靶点，还可抑制EGFR所在的ErbB受体家族的其他类型受体（ErbB1-4四个类型，也叫HER1-4四个类型），而且也是不管靶点有没有突变都能抑制。

二代EGFR-TKI的代表药物包括：阿法替尼、达克替尼（Dacomitinib）等。

第三代EGFR-TKI的作用特点为不可逆性、选择性抑制突变型EGFR。

也就是说，仅仅抑制突变的EGFR，不会抑制未突变的EGFR。

三代EGFR-TKI的代表药物包括：奥希替尼（AZD9291）、艾维替尼（avitinib）、Rociletinib（CO1686）、Olmutinib(BI1482694/HM61713)、ASP8274、Nazartinib （EGF816）、PF-06747775等。

EGFR突变肺癌病人使用一、二代EGFR-TKI靶向药平均治疗约9-14个月后会发生耐药。

纤细薯蓣皂苷诱导肺癌A549细胞自噬的作用及机制

·药学研究·纤细薯蓣皂苷诱导肺癌A 549细胞自噬的作用及机制Δ李燕 1＊，李亚梅 2，雷歌燕 1，康佳兰 3，刘明轩 1，张敏鸿 2，杨建琼 2 #（1.赣南医学院第一附属医院药学部，江西赣州　341000；2.赣南医学院第一附属医院临床医学研究中心，江西赣州　341000；3.泰和县人民医院药剂科，江西吉安　343700）中图分类号 R 965；R 285 文献标志码 A 文章编号 1001-0408（2024）08-0912-06DOI 10.6039/j.issn.1001-0408.2024.08.03摘要目的探讨吉祥草中纤细薯蓣皂苷（gracillin ）诱导人非小细胞肺癌A 549细胞自噬的作用及机制。

方法以A 549细胞为对象，采用CCK-8法检测不同浓度（0.25、0.5、1、2、4 μmol/L ）gracillin 作用不同时间（12、24、48 h ）对细胞增殖的影响。

与不加药物的对照组进行比较，采用生物透射电子显微镜观察gracillin （2 μmol/L ）作用24 h 对细胞自噬小体形成的影响；通过GFP-LC 3质粒转染实验检测gracillin （0.25、0.5、1、2 μmol/L ）作用24 h 后GFP-LC 3在细胞自噬小体膜上的聚集情况；采用实时定量聚合酶链式反应法和Western blot 法检测gracillin （0.25、0.5、1、2 μmol/L ）作用24 h 后A 549细胞中序列相似性家族102成员A （FAM 102A ） mRNA 和蛋白的表达水平，以及自噬相关蛋白[p 62、Beclin-1、微管相关蛋白1轻链3B （LC 3B ）]和磷脂酰肌醇3激酶（PI 3K ）/蛋白激酶B （又名Akt ）信号通路相关蛋白的表达水平。

结果 gracillin 对A 549细胞具有一定的增殖抑制作用，且呈浓度和时间依赖趋势；其作用24 h 的半数抑制浓度为2.55 μmol/L 。

国际上著名的从事药剂学研究的专家

Intra Oral Delivery (口腔内传递)直接由口腔黏膜吸收,瞬间进入血液循环,有效成分不流失。

Universities, Departments,FacultiesResearchersButler University College of Pharmacy and Health Sciences Health Sciences USA Associate Professor Nandita G. DasMain focus on her research facilities are about peformulation, biopharmaceutics, drug targeting, anticancer drug delivery.Purdue University School of Pharmacy and Pharmacal Sciences Department of Industrial and Physical Pharmacy (IPPH) USA Professor Kinam ParkControlled Drug Delivery, Glucose-Sensitive Hydrogels for Self-Regulated Insulin Delivery, Superporous Hydrogel Composites, Oral Vaccination using Hydrogel Microparticles, Fractal Analysis of Pharmaceutical Solid Materials.St. John's University School of Pharmacy and Allied Health ProfessionsUSA Professor Parshotam L. MadanControlled and targeted drug delivery systems; Bio-erodible polymers as drug delivery systemsThe University of Iowa College of Dentistry Department of Oral Pathology, Radiology, and Medicine USA Professor Christopher A. Squierpermeability of skin, and oral mucosa to exogenous substances, including alcohol and tobacco, and drug deliveryThe University of Iowa College of Pharmacy Department of Pharmaceutics USA Associate Professor Maureen D. DonovanMucosal drug delivery especially via the nasal, gastrointestinal and vaginal epithelia; and mechanisms of drug absorption and disposition.The University of Texas at San Antonio College of Engineering Department of Biomedical Engineering USA Professor Jeffrey Y. ThompsonDental restorative materials and implantsThe University of Utah Pharmaceutics & Pharmaceutical Chemistry USA Professor John W. MaugerDr. Maugner is mainly focused on dissolution testing and coating technology of orally administered drug products with bitter taste about which he is one of the inventors of a filed patent.University of Kentucky College of Pharmacy Pharmaceutical Sciences USA Professor Peter CrooksDr. Crooks is internationally known for his research work in drug discovery, delivery, and development, which includes drug design and synthesis, pharmacophore development, drug biotransformation studies, prodrug design, and medicinal plant natural product research. His research also focuses on preclinical drug development, including drug metabolism and pharmacokinetics in animal models, dosage form development, and drug delivery assessment using both conventional and non-conventional routes, and preformulation/formulation studies.Associate Professor Russell MumperDr. Mumper's main research areas are thin-films and mucoadhesive gels for (trans)mucosal delivery of drugs, microbicides, and mucosal vaccines, and nanotemplate engineering of nano-based detection devices and cell-specific nanoparticles for tumor and brain targeting, gene therapy and vaccines.West Virginia University School of Pharmacy Department of Basic Pharmaceutical Sciences USA Associate Professor Paula Jo Meyer StoutDr. Stout's research areas are composed of dispersed pharmaceutical systems, sterile product formulation DDS for dental diseases and coating of sustained release formulations.Monash University Victorian College of Pharmacy Department of Pharmaceutics Australia Professor Barrie C. FinninTransdermal Drug Delivery. Physicochemical Characterisation of Drug Candidates. Topical Drug Delivery. Drug uptake by the buccal mucosaProfessor Barry L. ReedTransdermal Drug Delivery. Topical Drug Delivery. Formulation of Dental Pharmaceuticals.University of Gent Faculty of Pharmaceutical Sciences Department of Pharmaceutics Belgium Professor Chris Vervaet-Extrusion/spheronisation - Bioadhesion - Controlled release based on hot stage extrusion technology - Freeze-drying - Tabletting and - GranulationPh.D. Els AdriaensMucosal drug delivery (Vaginal and ocular) Nasal BioadhesionUniversity of Gent Faculty of Pharmaceutical SciencesLaboratory of Pharmaceutical Technology Belgium Professor Jean Paul Remonbioadhesive carriers, mucosal delivery, Ocular bioerodible minitablets, Compaction of enteric-coated pellets; matrix-in-cylinder system for sustained drug delivery; formulation of solid dosage forms; In-line monitoring of a pharmaceutical blending process using FT-Raman spectroscopy; hot-melt extruded mini-matricesDanish University of Pharmaceutical Sciences Department of Pharmaceutics Denmark Associate Professor Jette JacobsenLow soluble drugs ?in vitro lymphatic absorption Drug delivery to the oral cavity ?in vitro models (cell culture, diffusion chamber) for permeatbility and toxicity of drugs, in vivo human perfusion model, different formulation approaces, e.g. iontophoresis.。

胰岛素抵抗的分子学机制

胰岛素抵抗的分子学机制李影;闫鹏【摘要】胰岛素受体底物(IRS)丝氨酸磷酸化是胰岛素信号转导通路中的一种时间依赖性生理反馈机制,代谢及炎症应激阻断其磷酸化进而导致胰岛素抵抗.诱导胰岛素抵抗的因素激活了包括抑制性κB激酶(IKKβ)、c-Jun氨基端激酶(JNK)、细胞外信号调节激酶、雷帕霉素靶蛋白通路和p70S6激酶在内的激酶,进而导致失控的IRS丝氨酸磷酸化.因此,这些激酶是抗胰岛素抵抗的潜在药物靶点,IKKβ/核因子κB 或JNK通路靶向治疗未来可能发展为糖尿病治疗方法.【期刊名称】《医学综述》【年(卷),期】2014(020)017【总页数】3页(P3122-3124)【关键词】胰岛素;胰岛素抵抗;2型糖尿病;分子生物学【作者】李影;闫鹏【作者单位】河南科技大学第一附属医院心血管内科,河南洛阳471003;河南科技大学第一附属医院心血管内科,河南洛阳471003【正文语种】中文【中图分类】R34胰岛素抵抗是肥胖的一个主要特征，是代谢综合征的一个核心部分，也是2型糖尿病、心血管疾病、肝脏疾病发展过程中的一个重要的病理生理因素[1]。

胰岛素抵抗是由营养超负荷,系统性脂肪酸过剩,脂肪组织的炎症,内质网应激,氧化应激和脂肪组织缺氧之间复杂的相互作用引起的[2]，该文重点阐述药理学和基因学的相关研究。

1 胰岛素受体底物1/2丝氨酸磷酸化和胰岛素抵抗胰岛素刺激的胰岛素受体底物1/2(insulin receptor substrates 1/2,IRS1/2)的酪氨酸磷酸化促进IRS1/2连接并激活磷脂酰肌醇3-激酶,进而激活下游信号蛋白激酶B、非典型蛋白激C和哺乳动物的雷帕霉素靶蛋白通路(mammalian target of rapammclin,mTOR)。

这些通路均参与胰岛素的合成代谢过程。

胰岛素除了促进IRS1酪氨酸磷酸化外，还引起IRS1一些位点的丝氨酸磷酸化。

IRS1的丝氨酸残基磷酸化对胰岛素信号兼有正性和负性调节作用，通过系统性而非单一位点的磷酸化来调节IRS1可以解释这种错综复杂的调节模式[3]。

bovine gamma-globulin 分子量

bovine gamma-globulin 分子量法国生物学家Brachet和英国生物学家Hammersen于1932年发现，当新鲜的晒干黄疸猪血清在高盐条件下析出时，可得到一种Globulin。

它被称为γ-S-Globulin，后来又被称为γ-Globulin。

这种蛋白质是由220个氨基酸组成的多肽链，其中含有15个半胱氨酸（cysteine）并且没有碳酸酐基。

它的分子量约为146 kDa（千道尔顿），具体数值会受到具体实验方法和测量条件的影响。

牛源γ-球蛋白是一种来源于牛血清的γ-球蛋白，牛血清是一种天然的高分子混合物，包含多种不同的蛋白质。

γ-球蛋白是其中最丰富的成分之一，它在血浆中占总蛋白质的15-25％。

γ-球蛋白的分子量可以通过多种方法进行测定。

一种常用的方法是凝胶过滤法（gel filtration）或者凝胶电泳法（gel electrophoresis）。

在凝胶过滤法中，蛋白质溶液被滤过尺寸合适的凝胶柱，不同分子量的蛋白质会以不同速度通过凝胶柱，从而分离出不同大小的蛋白质。

通过测量不同蛋白质的迁移速度，可以计算出γ-球蛋白的分子量。

另外一种常用的方法是质谱分析（mass spectrometry），质谱分析可以直接测量蛋白质中各个氨基酸残基的质量，从而得到蛋白质的分子量。

这种方法通常需要先将蛋白质进行裂解和鉴定，然后使用质谱分析仪器进行测量。

除了实验方法外，还可以通过基因组学和蛋白质组学的方法进行分子量的预测。

通过测量物种基因组中相应的基因或者利用已知的蛋白质序列，可以利用计算机算法预测蛋白质的分子量。

γ-球蛋白在免疫系统中起着重要的作用。

它主要由免疫球蛋白IgG组成，具有抗体的功能。

γ-球蛋白能够识别并结合特定的抗原，从而触发免疫反应。

在牛源γ-球蛋白中，IgG是主要的成分，它可以通过免疫电泳等方法进行分离和纯化。

γ-球蛋白有很多重要的应用，包括医学和生物技术领域。

在医学方面，γ-球蛋白可以用于治疗免疫缺陷病和自身免疫性疾病。

双氢青蒿素通过调节TGF-β

doi：10.3969/j.issn.1000-484X.2023.09.011双氢青蒿素通过调节TGF-β/Smad通路改善小鼠接触性皮炎①孙鸣远②金权鑫③张馨元张琪李芳芳金桂花（延边大学医学院免疫学与病原生物学教研室，延吉 133002）中图分类号R284.2 文献标志码 A 文章编号1000-484X（2023）09-1852-06［摘要］目的：探讨双氢青蒿素（DHA）对小鼠接触性皮炎（CHS）的抑制作用及机制。

方法：0.5%2，4-二硝基氟苯（DNFB）涂抹小鼠腹部连续2 d致敏，5 d后用0.25%DNFB涂抹左耳发敏，右耳涂抹丙酮和橄榄油混合液作为对照，于致敏前2 d灌胃给予DHA处理。

HE染色观察小鼠皮肤组织病理学变化，免疫组化染色观察小鼠耳部皮肤CD4+T、CD8+T细胞浸润情况，测定脾脏指数。

ELISA检测血清IL-6、IFN-γ、IL-10、TGF-β和单核细胞趋化蛋白（MCP）-1变化，Western blot检测皮肤Smad2和Smad3磷酸化水平，流式细胞术检测皮肤和脾脏免疫细胞浸润情况。

结果：DHA可显著改善CHS小鼠耳朵肿胀、皮肤红斑及脾脏指数（P<0.05）。

组织病理学结果显示，DHA处理可明显抑制CHS小鼠皮肤增厚和炎症细胞浸润。

流式细胞术结果显示，DHA处理后皮肤和脾脏中浸润的CD4+T细胞、CD8+T细胞、树突状细胞和巨噬细胞显著减少（P<0.05）。

ELISA结果显示，相对于模型组，DHA处理组血清IL-6、IFN-γ、TGF-β和MCP-1水平明显降低（P<0.05）。

Western blot结果表明DHA处理显著抑制皮肤Smad2和Smad3磷酸化水平（P<0.05）。

结论：DHA通过减少免疫细胞浸润和调节TGF-β/Smad信号传导抑制CHS，为治疗CHS提供了新的药物选择和实验依据。

［关键词］接触性皮炎；双氢青蒿素；TGF-β；Smad2/3Dihydroartemisinin ameliorates contact hypersensitivity in mice by regulating TGF-β/Smad signaling pathwaySUN Mingyuan， JIN Quanxin， ZHANG Xinyuan， ZHANG Qi， LI Fangfang， JIN Guihua. Department of Immunology and Pathogenic Biology， Yanbian University Medical College， Yanji 133002， China［Abstract］Objective：To investigate inhibition of dihydroartemisinin （DHA） on contact hypersensitivity （CHS） in mice and its mechanisms. Methods：Mice were sensitized with 0.5%2，4-dinitrofluorobenzene （DNFB）on shaved abdominal flank skin for 2 consecutive days， and CHS was elicited by 0.25%DNFB on left ear 5 days later. Right ear was treated with acetone/olive-oil alone as control. Mice were given DHA orally 2 days before sensitization. Skin histopathological changes were observed by HE staining，infiltra‐tions of CD4+T and CD8+T cells in ear skin were observed by immunohistochemical staining， and spleen index was detected. Serum IL-6，IFN-γ， IL-10， TGF-β and monocyte chemotactic protein （MCP）-1 changes were detected by ELISA， skin Smad2 and Smad3 phos‐phorylation levels were detected by Western blot，and skin and spleen immune cells infiltration were detected by flow cytometry. Results：DHA significantly improved ear swelling， skin erythema and spleen index of CHS mice （P<0.05）. Histopathological analysis revealed that degree of edema and cellular infiltration were markedly decreased in DHA-treated CHS mice. Flow cytometry results show that DHA treatment significantly decreased CD4+T cells， CD8+T cells， dendritic cells and macrophages infiltration in ear skin and spleen （P<0.05）. ELISA results showed that DHA treatment also diminished serum levels of IL-6， IFN-γ， TGF-β and MCP-1 compared with model group （P<0.05）. Western blot showed that Smad2 and Smad3 phosphorylation were normalized by DHA treat‐ment （P<0.05）. Conclusion：DHA suppresses CHS by reducing infiltration of immune cells and regulating TGF-β/Smad signaling pathway， which provides a new drug selection and experimental basis for CHS treatment.［Key words］Contact hypersensitivity；Dihydroartemisinin；TGF-β；Smad2/3①本文为国家自然科学基金项目（81960305）。

Ubiquitin-Proteasome Pathway

Ubiquitin-Proteasome Pathway
SIGMA-ALDRICH
Ubiquitin-Proteasome Pathway
Intracellular proteolytic systems recognize and destroy misfolded or damaged proteins, unassembled polypeptide chains, and short-lived regulatory proteins. There are several mechanisms for protein degradation within cells. Two systems that play important roles in proteolysis resulting from cell stress are the calpain proteases and the ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway functions widely in intracellular protein turnover. It plays a central role in degradation of short-lived and regulatory proteins important in a variety of basic cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen processing and presentation. The pathway employs an enzymatic cascade by which multiple ubiquitin molecules are covalently attached to the protein substrate. The polyubiquitin modification marks the protein for destruction and directs it to the 26S proteasome complex for degradation. References Sommer, T., et al., Compartment - specific functions of the ubiquitin - proteasome pathway. Rev. Physiol. Biochem. Pharmacol., 142, 97-160 (2001). Doskeland, A.P., and Flatmark, T., Cowith polyubiquitin chains catalysed by rat liver enzymes. Biochim. Biophys. Acta., 1547, 379-386 (2001). Hartmann-Petersen, R., et al., Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking. Arch. Biochem. Biophys., 386, 89-94 (2001). Brooks, P., et al., Subcellular localization of proteasomes and their regulatory complexes in mammalian cells. Biochem. J., 346, 155-161 (2000).

羟氯喹应用于生殖免疫领域的研究进展

Vol.41No.3Mar.2021上海交通大学学报（医学版）JOURNAL OF SHANGHAI JIAO TONG UNIVERSITY (MEDICAL SCIENCE)羟氯喹应用于生殖免疫领域的研究进展寇禧，赵爱民上海交通大学医学院附属仁济医院妇产科，上海200127［摘要］羟氯喹（hydroxychloroquine ，HCQ ）是人工合成的4-氨基喹啉类抗疟药。

近年来，其调节免疫、免疫抑制、抗炎、保护血管内皮、预防血栓、降糖调脂、抗感染等多种药理作用逐渐为人所知。

目前HCQ 已成为治疗系统性红斑狼疮的一线用药，多个自身免疫疾病诊治指南将其列为妊娠期及哺乳期可全程使用药物。

该文主要阐述了在生殖免疫领域包括妊娠合并系统性红斑狼疮、抗磷脂综合征、干燥综合征等自身免疫疾病，及不明原因习惯性流产和反复胚胎种植失败，应用HCQ 的机制以及相关研究的进展，探讨HCQ 应用于生殖免疫领域的安全性及前景。

［关键词］羟氯喹；生殖免疫；妊娠；药物机制；安全性［DOI ］10.3969/j.issn.1674-8115.2021.03.017［中图分类号］R714.259［文献标志码］AResearch progress of hydroxychloroquine in reproductive immunologyKOU Xi,ZHAO Ai -minDepartment of Obstetric and Gynecology,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China[Abstract ]Hydroxychloroquine (HCQ)is a kind of synthetic 4-aminoquinoline antimalarial drugs.In recent years,other pharmacological effects of HCQ,such as immunomodulatory,immunosuppression,anti-inflammation,vascular endothelium protection,anti-thrombus,improving metabolic syndrome,and anti-infection,have been gradually known.At present,HCQ is a first-line drug for systemic lupus erythematosus.And in many guidelines for the treatment of autoimmune diseases,HCQ is recommended during whole pregnancy and lactation.This review mainly expounds the mechanism and research progress of HCQ in reproductive immunology,including pregnancy complicated with autoimmune diseases,such as systemic lupus erythematosus,antiphospholipid syndrome and Sjogren's syndrome,as well as unexplained recurrent spontaneous abortion and recurrent implantation failure,and explores the safety and application prospect of HCQ in reproductive immunology.[Key words ]hydroxychloroquine (HCQ);reproductive immunology;pregnancy;drug mechanism;safety羟氯喹（hydroxychloroquine ，HCQ ）来源于抗疟药。

碧萝芷通过激活蛋白激酶b改善aml12细胞胰岛素抵抗

DOI：10.12007/j.issn.0258‐4646.2020.01.004
Pycnogenol improves insulin resistance by activating AKT expression in AML12 cells
ZHONG Wenxia1，WANG Yiying1，2，FAN Bin3，GU Jianqiu1
PYC10+OA组、PYC50+OA组培养基上清葡萄糖剩余含量明显减少（P < 0.05），糖原含量明显增加（P < 0.05）。 PYC10+OA组、
PYC50+OA组分别与OA组比较，糖异生基因G6PC、PEPCK、PGC1α mRNA 表达无统计学差异（P > 0.05），糖酵解基因Eno1、Gpi1
促进 PKLR表达增加糖酵解，并且促进p-GSK3β表达增加糖原合成来调控AML12细胞胰岛素抵抗。
关键词碧萝芷；胰岛素抵抗；糖异生；糖酵解
中图分类号 R587
文献标志码 A
文章编号 0258-4646（2020）01-0016-05
网络出版地址 /kcms/detail/21.1227.R.20191211.1413.006.html
摘要目的探讨碧萝芷（PYC）对油酸（OA）诱导的AML12细胞胰岛素抵抗的作用及其相关分子机制。方法将细胞分为空白
对照组、PYC50（50 µg/mL）组、OA（200 µmol/L）组、PYC10（10 µg/mL）+OA（200 µmol/L）组、PYC50（50 µg/mL）+OA（200 µmol/L）
1材料与方法11试剂油酸oleicacidoa购自美国sigma公司pyc购自瑞士horphag研究有限公司葡萄糖氧化酶试剂盒购自上海荣盛生物有限公司糖原染色periodicacidschiffstainpas试剂盒购自沈阳万类有限公司胰岛素受体底物irspirs抗体蛋白激酶baktpakt抗体磷酸化糖原合成酶激酶3pgsk3抗体gapdh抗体均购自美国cst公司实时pcr引物购自上海生工有限公司pcr试剂盒均购自日本takara公司

模式识别受体

① Toll-like receptors; TLRs ② RNA sensor（RIG-I, MDA5…） ③ DNA sensor（AIM2，DAI…） ④ NOD-like receptors； NLRs ⑤ C-Lectin receptors ⑥ Other PRRs
PRR-PAMPs engagement activates phagocytes
Ralph M. Steinman 1973，Ralph Steinman isolate Dentritic Cell)。 DC can activate T cell. DC is the birdge between innate immunity and adaptive immunity.
University of Texas Southwestern HHMI
Prion-like protein fight off viruses: MAVS, Cell 2011
2-3）RIG-1/MDA-5 signaling pathway
2-4） regulator of RIG-I/MDA-5
Toll Pathway
Drosophila
They all deserve the Nobel Prize
Charles Janeway 1943–2003
Yale Univ. PRR
Ruslan Medzhitov Yale Univ. TLR
Takashi Fujita, Shizuo Akira Kyoto Univ., Osaka Univ.
LPS Flagellin 。。。。
pattern recognition receptors, PRR：
Receptors, on the surface or inside the cells, for those pathogen associated molecule patterns.

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分子量486.59
溶解性（25°C）
DMSO 44 mg/mL
分子式C H N O S Water <1 mg/mL
CAS号1353550-13-6Ethanol <1 mg/mL
储存条件3年 -20°C 粉末状
生物活性
BTK is a member of the Tec family of non-receptor protein tyrosine kinases. BTK is mostly expressed in hematopoietic cells such as B cells, mast cells and macrophages. BTK plays key roles in multiple cell signaling pathways including B-Cell Receptor (BCR) and Fc receptor (FcR) signaling cascades and is an essential mediator not only in B-cell dependent but also in myeloid cell dependent inﬂammatory arthritis. HM71224 has been selected as a novel therapeutic agent for the treatment of autoimmune diseases such as rheumatoid arthritis (RA).
不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）
小鼠大鼠兔豚鼠仓鼠狗
重量 (kg)0.020.15 1.80.40.0810
体表面积 (m)0.0070.0250.150.050.020.5
K系数36128520
动物 A (mg/kg) = 动物 B (mg/kg) ×
动物 B的K系数
动物 A的K系数
例如，依据体表面积折算法，将白藜芦醇用于小鼠的剂量22.4 mg/kg 换算成大鼠的剂量，需要将22.4 mg/kg 乘以小鼠的K系数（3），再除以大鼠的K系数（6），得到白藜芦醇用于大鼠的等效剂量为11.2 mg/kg。

Olmutinib 目录号M5133
化学数据
262662
2
m
m
m
m m。