【作者】 王召华；

【导师】 钱叶本；

【作者基本信息】 安徽医科大学 ， 外科学， 2011， 硕士

【摘要】 目的:探讨大鼠肝脏缺血再灌注损伤(I/R)后胆汁酸转运体Bsep与Mrp2的表达及意义方法:健康的雄性Sprague-Dawley(S-D)大鼠60只随机分为两组(每组30只):A组为对照组,进腹后仅解剖第一肝门,不行第一肝门阻断;B组为缺血再灌注组,解剖第一肝门,并用沙氏钳夹闭第一肝门,造成门静脉、肝动脉和胆总管的完全阻断,阻断30min后开放,恢复入肝血流。分别于术后1h、6h、12h、1d、3d开腹,每个预定时相点取5只大鼠进行取材和指标检测,应用全自动生化分析仪检测血清ALT、DBIL、TBA的含量,应用HE染色方法检测各组大鼠肝组织病理学改变,应用免疫组织化学SP(Streptavidin-perosidase)法和实时定量逆转录-聚合酶链反应(Real time Reverse Transcriptase-PCR)技术,检测各组大鼠肝组织中的Bsep与Mrp2蛋白和mRNA表达情况。结果:①TBA、DBIL、ALT:A组大鼠各时相点血清TBA、DBIL、ALT含量无明显差别(P>0.05)。B组大鼠缺血再灌注后1h、6h、12h、1d血清中TBA、DBIL、ALT的含量明显升高,较之A组各对应时相点具有明显差别(P<0.05);再灌注后3d,血清中TBA、DBIL、ALT含量与A组比较无明显差别(P>0.05)。②病理学改变:A组大鼠术后1h-3d肝细胞形态正常,均无肝细胞坏死。B组大鼠缺血再灌注术后1h改变较轻,肝细胞轻度水肿;再灌注术后6h、12h、1d、3d可见肝细胞水肿明显,脂质沉积,细胞核增大、稍深染,并可见核分裂相,但各时相点均未见肝细胞坏死的发生。③RT-PCR结果:A组大鼠术后1h、6h、12h、1d、3d各时相点肝组织中Bsep mRNA的含量无明显差别(P>0.05);B组大鼠缺血再灌注术后1h、6h、12h、1d,肝组织中Bsep mRNA含量减少,与A组各对应时相点比较有显著差别(P<0.05),术后3d两组肝组织中Bsep mRNA的表达无显著差别(P>0.05)。A组大鼠术后1h、6h、12h、1d、3d各时相点肝组织中Mrp2 mRNA的含量无明显差别(P>0.05);B组大鼠缺血再灌注术后1h、6h、12h,肝组织中Mrp2 mRNA含量减少,与A组各对应时相点比较有显著差别(P<0.05),术后1d、3d两组肝组织中Mrp2 mRNA的表达无明显差别(P<0.05)。④免疫组化结果:A组大鼠在术后的1h、6h、12h、1d、3d,肝组织中Bsep、Mrp2蛋白在肝细胞膜上规则表达,显露出肝细胞轮廓,胞浆内未见棕黄染色。B组大鼠在缺血再灌注后的1h、6h、12h、1d,肝组织中Bsep蛋白在肝细胞膜上表达不规则,染色稀疏,与A组对应各时相点比较明显下调,具有统计学意义(X~2=7.33、X~2=10.00、X~2=10.00和X~2=7.00,P<0.05),以术后6h下调最为显著(P<0.01),且胞浆内均未见棕黄染色;再灌注后3d,Bsep蛋白在两组中的表达无明显差别(X~2=2.50,P>0.05)。B组大鼠在缺血再灌注后的1h、6h、12h,1d,肝组织中Mrp2蛋白在肝细胞膜上表达稀疏,散乱,并可见胞浆内棕黄染色,与A组对应各时相点比较明显下调,具有统计学意义(X~2=10.00、X~2=10.00、X~2=10.00和X~2=7.00,P<0.05);同时胞浆内可见棕黄染色,Mrp2蛋白在胞浆内散在分布,与对应A组各时相点比较有显著差别(X~2=10.00、X~2=8.00、X~2=6.67和X~2=8.00,P<0.05),再灌注后3d,两组肝组织中Mrp2蛋白重新表达分布在肝细胞膜上,显露肝细胞轮廓,在两组中的表达无明显差别(X~2=0.40,P>0.05)。结论:大鼠肝脏缺血再灌注损伤(I/R)后肝组织中Bsep表达下调和Mrp2定位异常可能是鼠肝缺血再灌注损伤后高胆红素血症发生的重要机制。更多还原

【Abstract】 Objective: To detect the expression and location of Bsep and Mrp2 after hepatic ischemia-reperfusion: experiment with rats,and to investigate the mechanism of hyperbilirubinemia after rat hepatic ischemia-reperfusion.Methods: Sixty healthy MaleSprague-Dawley(S-D) rats were randomly divided into 2 groups(thirty ras in each group):Group A(the control group),rats were not subjected to hepatic ischemia reperfusion.Group B,rats were subjected to 30 min of total hepatic ischemia.At 1h,6h,12h,1d,3d after operation checkpoint was set. Routine biochemistry method detect TBA,DBIL, ALT levels of plasm.The expression of Bsep and Mrp2 mRNA was determined by Real Time reverse transcriptase PCR .The expression of Bsep and Mrp2 protein was detected by immunohistochemical SP method .Results:①TBA、DBIL、ALT:There were no significant differents in levels of serum TBA、DBIL、ALT in control group from 1h-3d after operation(P>0.05)。In group B the expression of TBA、DBIL、ALT were significantly higher compared with group A at 1h,6h,12h,1d after hepatic ischemia-reperfusion(P<0.05),and on significant differences at 3d(P>0.05).②HE:Pathological examination showed that there were only amid inflammation in the liver tissues that had undergone ischemia-reperfusion in Group B,and no necrosis of hepatocytes was seen in both group.③RT-PCR:There were no significant differences in the levels of serum Bsep and Mrp2 from 1h-3d in Group A(P>0.05). The expressions of serum Bsep at 1h,6h,12h,1d after hepatic ischemia-reperfusion were significant differences compared with Group A(P<0.05),and there was no significant differences at 3d(P>0.05).At 1h,6h,12h after hepatic ischemia-reperfusion in rats of Group B were significant lower than Group A(P<0.05),and there were no significant at 1d,3d.(P>0.05).④IHC:The protein of Bsep and Mrp2 were regularly expressed in hepatocyte membrane in both group. At 1h-1d after reperfusion ,the protein of Bsep was significant derease in Group B compared with Group A,no location in hyalomitome,and there were no significan at 3d. The localization of Mrp2 continuously altered at 1h-1d,not only expression in hepatocyte membrane,but also can be seen in hyalomitome,and there were no significan at 3d.Conclusion: Down-regulation of Bsep and absence of Mrp2 localization in hepatocytes membrane may be the mechanism of serum bilirubin after hepatic ischemia-reperfusion.更多还原