摘要
目的研究长时间冷缺血对供肝胆管细胞原纤毛结构及功能的影响及其与移植肝胆管病的相关性。方法选取6~8周龄雄性同基因近交系Wistar大鼠60只,随机分为实验组(供肝冷缺血时间为12h)与对照组(供肝冷缺血时间为1h),每组30只。两袖套法建立大鼠肝移植模型,采用改良Gao支架法重建肝动脉血供,支架法重建胆管。术后2、8、16周分别活杀5只大鼠,抽静脉血查血生化指标,切取肝脏标本分别用福尔马林、2.5%戊二醛及液氮固定,观察肝组织形态学改变及肝内胆管细胞及原纤毛的变化,免疫荧光法和免疫印迹分析法检测胆管细胞原纤毛α-tubulin、膜蛋白(PC-1、TRPV4及P2Y12)的表达。结果与对照组相比较,实验组汇管区胆管结构模糊或胆管消失,小叶间胆管部分上皮细胞脱落,胆管壁内胶原暴露,周围有较多的胶原沉积和炎细胞浸润,胆管壁内胶原暴露,部分胆管上皮细胞变形、坏死,原纤毛及微绒毛减少或消失。原纤毛α-tubulin、PC-1、TRPV4及P2Y12的表达逐渐减少。结论供肝长时间冷缺血可导致胆管细胞原纤毛结构及功能的缺损,与移植肝胆管病的发生具有显著的相关性。
Objective To study the impaired cholangiocytes primary cilia caused by long-term cold ischemia and to investigate its correlation with graft cholangiopathies after orthotopic liver transplantation(OLT).Methods Sixty male Wistar rats aged 6-8 weeks were divided randomly into 2 groups:control group with 1-hour donor liver cold ischemia(n=30) and study group with 12-hour donor liver ischemia(n=30).Two-cuff method was used to establish an OLT model,and the hepatic artery and bile duct were reconstructed by stents.All samples were harvested for testing in 2,8 and 16 weeks after the operation,and in each group 5 mice were tested on indicated time point.The serum biochemical indicators were measured.The morphological changes of intrahepatic bile ducts and cholangiocytes primary cilia were observed by an optical microscope and an scanning electronic microscope respectively.The ciliary marker(α-tubullin) and membrane proteins(PC-1,TRPV4 and P2Y12) were detected by immunofluorescence analysis and Western blot respectively.Results In the study group,phlogocytes infiltrated and bile ducts proliferated remarkably at 8 weeks.At 16 weeks,the biliary structures were vague and some bile ducts disappeared,with collagen deposition and phlogocytes infiltration around the ducts,and some biliary epithelial cells(BECs) deformed or necrosed and primary cilia disappeared.In the control group,the structures of intrahepatic bile ducts and BECs were almost integrated and the primary cilia were present.In the study group,the expressions of α-tubulin,PC-1,TRPV4 and P2Y12 in the bile ducts disappeared after 8 weeks,while in the control group,they decreased at 2 weeks and increased slightly after 8 weeks.Conclusions Donor liver long-term cold ischemia could cause graft cholangiopathies by impairing the integrity and functions of cholangiocytes primary cilia.
出处
《中华普外科手术学杂志(电子版)》
2012年第4期20-23,共4页
Chinese Journal of Operative Procedures of General Surgery(Electronic Edition)
基金
国家自然科学基金资助项目(81170454)
国家863计划课题项目(2001AA020103)
关键词
肝移植
冷缺血
再灌注损伤
细胞结构
移植肝胆管病
Liver transplantation
Cold ischemia
Reperfusion injury
Cellular stuructures
Graft cholangiopathies