bcl-2在mitoKATP通道开放剂二氮嗪诱导的抗小鼠移植肝脏缺血再灌注损伤中的作用的研究  

An Experiment Study on the Role of bcl-2 in Attenuation Ischemia-reperfusion Injury in Liver Graft in Mice Induced by mitoKATP Channel Opener Diazoxide

作　　者：吴乔[1] 别平[1] 唐春[2] 张玉君[1] 

机构地区：[1]第三军医大学西南医院全军肝胆外科研究所中国人民解放军西南肝胆外科医院,重庆400038 [2]第三军医大学大坪医院肝胆外科,重庆400042

出　　处：《实用临床医药杂志》2008年第2期35-40,共6页Journal of Clinical Medicine in Practice

基　　金：国家自然科学基金资助项目(30571806)

摘　　要：目的研究bcl-2在线粒体ATP敏感性钾通道(mitoKATP)开放剂二氮嗪(DZ)诱导的抗小鼠移植肝脏缺血再灌注损伤中的作用,探讨应用二氮嗪减轻移植肝缺血再灌注损伤的可行性。方法两袖套法建立小鼠同种异体原位肝移植模型,利用化学合成bcl-2基因的SiRNA,经门静脉高压注射转染供体肝脏。随机分为肝脏移植组、SiRNA-bcl-2转染移植组、DZ灌注移植组、DZ灌注+SiRNA-bcl-2转染移植组、阴性对照组。肝脏移植术后第3天用萤光定量PCR和Western-Blot观察各组肝组织的bcl-2 mRNA和蛋白表达变化;测定受体血清AST、ALT含量。TUN EL法检测肝细胞凋亡。结果二氮嗪灌注组bcl-2 mRNA和蛋白表达水平较肝移植组明显增高(P<0.01),bcl-2mRNA和蛋白表达分别增加(63.2±4.8)%和(83.5±6.4)%;bcl-2 SiRNA转染组bcl-2 mRNA和蛋白表达水平较肝移植组明显降低(P<0.01),bcl-2mRNA和蛋白表达抑制分别为(65.7±5.3)%和(83.4±6.3)%;DZ灌注+SiRNA转染移植组bcl-2mRNA和蛋白表达与肝移植组和阴性对照组比较无显著差异(P>0.05);二氮嗪(DZ)灌注组中肝细胞凋亡指数、血清AST、ALT含量明显低于各组。bcl-2 SiRNA转染移植组中肝细胞凋亡指数、血清AST、ALT含量明显高于各组。结论bcl-2基因表达上调在线粒体ATP敏感性钾通道开放剂二氮嗪诱导的抗小鼠移植肝脏缺血再灌注损伤中起重要作用,二氮嗪能通过诱导bcl-2基因表达减轻移植肝缺血再灌注损伤。Objective To explore the effect of bcl-2 in attenuation ischemia-reperfusion injury （I/RI） in liver graft in mice induced by mitochondrial ATP-sensitive potassium channel （mitoKATP） opener diazoxide and the feasibility of reducing ischemia reperfusion injury of graft liver by diazoxide. Methods Experiments of orthotopic liver transplantation were performed by two-cuff method. In vivo delivery of SiRNA targeting bd-2 mRNA into liver in donator was performed via the portal vein by high-volume injection. Mice were randomly divided into five groups： the liver transplantation group; SiRNA-bd-2 transfection group, diazoxide （DZ） perfusion group, DZ perfusion and SiRNA-bcl-2 transfection group and non-sense SiRNA transfection group. 3 days after liver transplantation, the expression of bcl-2 gene and protein level were determined by RT-PGR and Western blot. The serum AST and ALT level was detected. Cell apoptosis were observed by TUNEL.Results Diazoxide significantly improved the expression of bcl-2 in DZ perfusion group both at rnRNA and protein levels. （63.2 ± 5.3） % and （83.5 ± 6.4） %, respectively） ; bcl-2 SiRNA sig- nificantly inhibited the expression of bcl-2 in SiRNA-bcl-2 transfection group both at mRNA and protein levels （65.7 ± 4.8） % and （83.4 ± 6.3） %, respectively; the difference between liver transplantation group or DZ perfusion and SiRNA-bcl-2 transfection group or non-sense SiRNA transfection group is not significant. The proportion of hepatocyte apoptosis and the serum AST, ALT level in diazoxide perfusion group were lower than that in the other groups, while, proportion of hepatocyte apoptosis and the serum AST, ALT level in SiRNA-bcl-2 transfection group were higher than that in the other groups. Conclusion The up-regulation of bcl-2 gene expression plays an important role in against ischemia-reperfusion injury in graft liver in mice by mitoKATP opener diazoxide. Diazoxide can attenuate I/R I in graft liver by enhancing the expression of bcl-2.

关 键 词：肝移植 缺血再灌注损伤 线粒体ATP敏感性钾通道 二氮嗪 BCL-2 RNA 小干扰 

分 类 号：R657.3[医药卫生—外科学]