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目的探讨鼠肝缺血再灌注后胆汁淤积发生的分子机制。方法应用70%的鼠肝缺血35 min再灌注模型,检测胆汁、血浆中胆红素、胆酸的含量；荧光定量聚合酶链反应(PCR)、免疫组织化学方法分析毛细胆管膜上胆盐输出泵(Bsep)、多耐药相关蛋白2(Mrp2)的表达；激光共聚焦方法分析Mrp2定位的改变。结果再灌注后6 h、1、3 d,Bsep的表达明显下调(mRNA表达水平分别为0．189±0．044、0．240±0．078、0．224±0．068),与胆汁、血浆中胆酸的异常改变一致。Mrp2表达的明显下调仅发生于再灌注后的6h(mRNA表达水平为0．038±0．032),与再灌注后1 h～5 d胆红素的异常变化不相符。再灌注后6h—5d,Mrp2在毛细胆管膜上定位减少、向胞浆内分布。结论Bsep表达的减少以及Mrp2定位的异常是鼠肝缺血再灌注后胆汁淤积发生的主要分子机制。
Objective To investigate the molecular mechanism of cholestasis after ischemia-reperfusion in rat liver. Methods 70% rat liver ischemia reperfusion model was used to detect the content of bilirubin and bile acid in the bile and plasma. Fluorescent quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to analyze the bile salts (Bsep) and multidrug resistance-associated protein 2 (Mrp2) were detected by laser scanning confocal microscopy. Laser scanning confocal microscopy was used to analyze the localization of Mrp2. Results The expression of Bsep was significantly down-regulated at 6 h, 1 and 3 d after reperfusion (mRNA levels were 0.189 ± 0.044,0.240 ± 0.078 and 0.224 ± 0.068, respectively) , The abnormal change of cholic acid in plasma is consistent. Significant downregulation of Mrp2 expression occurred only 6h after reperfusion (mRNA expression level was 0.038 ± 0.032), which was not consistent with abnormal changes of bilirubin 1 h to 5 d after reperfusion. 6h-5d after reperfusion, Mrp2 localized in the bile duct membrane to reduce the distribution to the cytoplasm. Conclusion The decrease of Bsep expression and Mrp2 localization are the main molecular mechanisms of cholestasis after ischemia-reperfusion in rat liver.