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目的了解大鼠肝外胆管缺血再灌注损伤后成纤维细胞的凋亡和增殖情况及其相关基因表达的时序性变化。方法检测缺血大鼠肝外胆管再灌注后1、3、24、72 h肝外胆管壁成纤维细胞的凋亡指数(TUNEL标记法)、Fas和增殖细胞核抗原(PCNA)蛋白的表达状况(免疫组织化学法)以及Bcl-2 mRNA的表达状况(原位杂交法)。结果缺血再灌注后1 h,成纤维细胞的凋亡指数以及Fas、PCNA和Bcl-2 mRNA表达与对照组无明显差异。再灌注后3 h,成纤维细胞凋亡指数升高,24 h时达到高峰,此后逐渐下降。Fas蛋白表达与凋亡同步;PCNA表达在再灌注后3 h时已明显增强,接近峰值水平,此后一直维持较高水平。再灌注3 h时Bcl-2 mRNA表达与对照组无明显差异,24 h迅速升高并达到峰值,此后逐渐下降。结论肝外胆管缺血再灌注早期成纤维细胞有一定程度的凋亡,但随后减弱,其增殖在早期即明显加强,且一直保持到再灌注后期。
Objective To investigate the apoptosis and proliferation of fibroblasts after ischemia / reperfusion injury in rat extrahepatic bile duct and the temporal changes of the expression of related genes. Methods The apoptosis index (TUNEL), expression of Fas and proliferating cell nuclear antigen (PCNA) in extrahepatic bile duct fibroblasts were detected at 1, 3, 24, 72 h after reperfusion of extrahepatic bile duct in rats Immunohistochemistry) and the expression of Bcl-2 mRNA (in situ hybridization). Results The apoptosis index of fibroblasts and the expression of Fas, PCNA and Bcl-2 mRNA in fibroblasts at 1 h after ischemia-reperfusion were not significantly different from those in control group. At 3 h after reperfusion, the apoptotic index of fibroblasts increased and peaked at 24 h, then decreased gradually. The expression of Fas protein was synchronized with apoptosis. The expression of PCNA was significantly increased 3 h after reperfusion, close to the peak level, and remained high afterwards. At 3 h after reperfusion, the expression of Bcl-2 mRNA was not significantly different from that of the control group. The expression of Bcl-2 mRNA increased rapidly and peaked at 24 h, then decreased gradually. Conclusion The fibroblasts in the early stage of extrahepatic bile duct ischemia-reperfusion have a certain degree of apoptosis, but then weakened. The proliferation of the fibroblasts in the early stage is obviously enhanced and maintained till the later stage of reperfusion.