鼠肝缺血再灌注后血浆胆红素升高的分子机制

目的探讨鼠肝缺血再灌注后血浆胆红素升高的分子机制。方法实验分为假手术组、70%的鼠肝缺血20 min再灌注组和缺血35 min再灌注组,研究时点为再灌注后的6h、1d、3d和5d。HE染色分析肝组织病理改变。常规生物化学方法检测血浆及缺血肝叶胆汁中胆红素含量的变化。RT-PCR检测肝组织多药耐药相关蛋白2（MRP2）的表达。免疫组织化学方法分析MRP2在肝细胞毛细胆管膜上的定位。结果缺血20 min组和35 min组再灌注模型炎症反应轻,无肝细胞坏死的发生。与假手术组比较,缺血20 min组血浆中胆红素含量的升高、胆汁中胆红素含量的下降发生于再灌注后的6h～1d;缺血35 min组却持续至再灌注后的3～5 d。RT-PCR发现,缺血20 min组和缺血35 min组MRP2 mRNA表达的明显下调仅发生于再灌注后的6 h。免疫组织化学法发现缺血35 min组MRP2在胞膜下呈“囊状”分布,在毛细胆管膜上的定位减少。结论MRP2在毛细胆管膜上的定位减少很可能是鼠肝缺血再灌注后血浆胆红素升高发生的分子机制。

The mechanism of the increase of plasma bilirubin after hepatic ischemia-reperfusion in rats

OBJECTIVE: To investigate the mechanism of plasma bilirubin level increase after hepatic ischemia-reperfusion in rats. METHODS: Rats were divided into a sham operation group (A group), a 20 min ischemia-reperfusion group (B group) and a 35 min ischemia-reperfusion group (C group). Study time points were 6 hours and 1, 3, and 5 days after the reperfusion. Pathological changes in the livers were studied with histological slides stained with hematoxilin and eosin. Routine biochemistry methods were used to detect the bilirubin level of blood plasma and the bile drained from the ischemic hepatic lobes. RT-PCR was used to analyze the expression of the multidrug resistance-associated protein 2 (MRP2) and mRNA. Immunohistochemistry was used to analyze the localization of MRP2 in the canalicular membrane. RESULTS: B and C groups showed a mild inflammatory reaction without hepatocyte necrosis. At 6 h and 1 day after reperfusion, there was a significant increase of the plasma bilirubin level and a decrease of the bilirubin level of the drained bile in B group. These changes lasted to the day 3 and day 5 in C group. MRP2 mRNA down-regulation was found at 6 h only in the B and C groups. No localization of MRP2 in the canalicular membrane was found but it appeared in "esicules" under the canalicular membrane in C group. CONCLUSIONS: Absence of MRP2 localization in the canalicular membrane could be the cause of the blood plasma bilirubin level increase after liver ischemia-reperfusion.