冷保存-再灌注损伤对大鼠移植肝胆管上皮细胞凋亡及bcl-2/bax表达的影响  

Effect of cold preservation and reperfusion injury on the apoptosis of hepatobiliary epithelial cells and bcl-2/bax gene expression in rats

作　　者：陈莉萍[1] 李州利[1] 戴睿武[2] 钱叶勇[1] 石炳毅[1] 

机构地区：[1]解放军总医院第二附属医院器官移植中心,北京市100091 [2]解放军成都军区总医院普通外科,四川省成都市610083

出　　处：《中国组织工程研究与临床康复》2009年第5期857-861,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：全军医学“十五”计划重大项目基金资助课题(06G115)~~

摘　　要：背景:移植肝胆管上皮细胞凋亡是影响肝移植后胆道功能恢复的因素之一,调控基因bcl-2/bax可能对胆管上皮细胞的生存起到决定作用。目的:观察缺血-再灌注损伤后大鼠肝内胆管上皮细胞的凋亡及调控基因bcl-2/baxmRNA表达的变化。设计、时间及地点:动物实验,细胞形态学观察,于2008-02/08在解放军第三军医大学西南医院肝胆外科研究所实验室完成。材料:健康雄性Wistar大鼠100只,随机分为3组:供肝冷保存1h组40只,冷保存12h组40只,对照组20只。方法:供肝冷保存1,12h组,组内随机配对,体质量相对较轻的大鼠做为供体,供肝置于4℃器官保存液中,分别保存1、12h后行原位肝移植,对照组只行开、关腹手术。在"两套袖法"基础上,以"支架法"建立动脉化大鼠原位肝移植模型,供受体肝总动脉采用改良"支架法"进行端端吻合,重建肝动脉血供。主要观察指标:分别于移植后1,3,7,14d检测血清总胆红素、碱性磷酸酶及肝内胆管上皮细胞的凋亡,实时荧光定量RT-PCR法检测胆管上皮细胞内bcl-2mRNA和baxmRNA的表达。结果:保存1h组移植后1,3d可见轻度胆道损伤的血清学及病理学表现,肝内胆管上皮细胞凋亡指数分别为(4.62±0.23)%,(3.42±0.22)%,(2.91±0.23)%,(2.87±0.16)%,且在移植后7d即与对照组无明显差异。在保存12h组移植后1,3,7d,胆汁郁积征象明显,伴有严重的胆管损伤病理学改变,且肝内胆管上皮细胞凋亡指数明显高于保存1h组和对照组,分别为(6.51±0.33)%,(8.52±0.36)%,(3.51±0.27)%,(2.91±0.28)%。在移植后各时相点,保存1h组肝内胆管上皮细胞内bcl-2mRNA/baxmRNA比值分别为(1.12±0.12)%,(1.34±0.13)%,(1.51±0.14)%,(1.60±0.15)%,在移植后7d即接近对照组水平,而保存12h组则为(0.90±0.08)%,(0.79±0.02)%,(1.36±0.12)%,(1.59±0.14)%,在移植后7d仍明显低于保存1h组和对照组,且与肝内胆管上皮细胞凋亡指数显著负相关(P=0.029)。结论:冷保存时间延�BACKGROUND： Cholangiocytes apoptosis in the grafts liver is an influencing factor of hepatobiliary recovery and the regulatory gene bcl-2/bax might play a key role in cholangiocytes surviving. OBJECTIVE： To investigate the expression of bcl-2/baxgene on the hepatobiliary epithelial cells （HEC） and apoptosis of HEC following ischemia/reperfusion injury. DESIGN, TIME AND SETTING： Animal experiment of cell morphology observation was performed at the Department of Hepatobiliary Surgery, Southwest Hospital, and the Third Military Medical University from February to August 2008. MATERIALS： One hundred healthy, male, Wistar rats. According to the cold preservation time of supplied liver, animals were randomly divided into the 1 hour cold preservation （CP1 h, n=-40）, 12 hours cold preservation （CP12 h, n=40） and the control （n=20） groups. METHODS： Rats in the preservation group were served as donor and recipients. The donor liver were preserved at UW solution at 4 ℃ for 1 or 12 hours and transplanted to the recipients. Based on the classic ＂two cuff＂ technique for orthotopic liver transplantation （OLT）, end-to-end anastomosis between the common hepatic artery of donor and recipient by the modified ＂Gao stent＂ was performed to reconstruct hepatic artery blood flow. MAIN OUTCOME MEASURES： The serum total bilirubin, alkaline phosphatase, apoptosis of the HEC, as well as the expression of bcl-2 mRNA and bax mRNA were studied at 1,3, 7, 14 days after transplantation. RESULTS： The CPlh group demonstrated slight signs of bile duct injury and low level of HEC apoptosis at 1 and 3 days after transplantation. Apoptosis index of HEC at each time point were （4.62±0.23）%, （3.42±0.22）%, （2.91±0.23）%, and （2.87±0.16）%, which was close to the control level at 7 days. While, the apoptosis index of HEC apoptosis index of HEC were （6.51±0.33） %, （8.52±0.36）%, （3.51±0.27）%, and （2.91±0.28）%. In CP1 h group, expression of bcl-2/bax mRNA in the HEC

关 键 词：原位肝移植 冷保存 再灌注 胆管上皮细胞 凋亡 

分 类 号：R657.3[医药卫生—外科学]