大鼠肝脏缺血再灌注损伤后胆汁酸转运体Bsep与Mrp2的表达及意义

摘要

Objective To investigate the mechanism of hyperbilirubinemia after rat hepatic ischemia-reperfusion. Methods Sixty healthy Male Sprague-Dawley(S-D) rats were randomly divided into 2 groups (thirty rag in each group) : Group A (the control group), rats were not subjected to hepatic ischemia reperfusion. Croup B, rats were subjected to 30 min of total hepaticischemia. At 1 h, 6 h, 12 h, 1 d, 3 d 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. Result Pathological examination showed that there were only amid inflammation in the liver tissues that had undergone ischemia-reperfusion in Croup B, and no necrosis of hepatocytes was seen in both group. In group B, the expression of TBA, DBIL, ALT were significantly higher compared with group A at 1 h -1 d after hepatic ischemia-reperfusion (P <0.05). The expressions of serum Bsep mRNA at 1 h - 1 d after hepatic ischemia-reperfusion were significant differences compared with Group A(P <0.05) , and the protein of Bsep expressed in hepatocyte membrane was significant derease in Croup B, and no location in hyalomitome. At 1 h ~ 12 h after hepatic ischemia-reperfusion in rats of Group B were significant lower than Group A(P< 0.05 ) , and there were no significant at 1 d( P > 0. 05 ). The localization of Mrp2 continuously altered at 1 h -1 d, not only expression in hepatocyte membrane, but also can be seen in hyalomitome. Conclusion Down-regulation of Bsep and absence of Mrp2 localization in hepatocytes membrane may be the mechanism of high level of serum bilirubin after hepatic ischemia-reperfusion.%目的 探讨大鼠肝缺血再灌注损伤(I/R)后高胆红素血症发生的分子机制.方法 60只健康雄性SD大鼠随机分成两组,A组为对照组,只解剖第一肝门,不行肝门阻断,B组为缺血再灌注后,阻断第一肝门30 min后开放.于术后的1h、6h、12 h、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TBA、DBIL、ALT的含量,应用HE染色方法检测各组肝组织病理学改变,应用免疫组织化SP(Streptavidin-perosidase)法和实时定量逆转录-聚合酶链反应(Read 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 鼠肝缺血30 min再灌注模型肝组织呈轻度炎症改变,各时相点均未见肝细胞坏死的发生.B组大鼠缺血再灌注后1h～1d血清中TBA、DBIL、ALT的含量明显增加,具有统计学意义(P＜0.05).B组大鼠缺血再灌注后1h～1d胆汁酸转运体Bsep mRNA表达水平明显下调(P＜0.05),同时Bsep蛋白在肝细胞膜上染色不规则、稀疏,蛋白质的表达减少,胞浆内未见表达；再灌注后1 h～12h,Mrp2 mRNA表达水平下调(P＜0.05),再灌注后1d,Mrp2 mRNA表达水平恢复正常,而再灌注后的1 h～1d,Mrp2蛋白在肝细胞膜上染色稀疏、散乱,表达减少,并可见胞浆内表达.结论 大鼠缺血再灌注损伤(I/R)后肝脏组织中Bsep表达下调和Mrp2定位改变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.