S100A6对小鼠原代肝星状细胞活化影响的研究

Effect of S100A6 on activation of hepatic stellate cells in mice

目的观察S100A6在肝组织中的表达并研究其在肝星状细胞活化中的作用。方法12只C57BL/6小鼠分为正常组及肝纤维化模型组,肝纤维化模型组以四氯化碳(CCl4)喂养6周复制肝纤维化模型,取肝脏行Masson染色评估纤维化程度,并采用免疫组化检测S100A6在肝组织中的表达,RT-qPCR和Western blot检测S100A6在肝组织中的mRNA和蛋白表达水平;提取C57BL/6小鼠原代肝星状细胞(hepatic stellate cells,HSCs)行体外培养,观察其在静止期和活化期的形态变化,以RT-qPCR和Western blot检测不同培养时间点S100A6 mRNA及蛋白表达;采用siRNA干扰原代HSCs S100A6后以RT-qPCR和Western blot检测S100A6、Ⅰ型胶原和α-SMA mRNA和蛋白表达。结果在CCl4诱导的小鼠肝纤维化组织中,Masson染色可观察其纤维化,而免疫组化亦可见S100A6明显表达于肝纤维化组肝脏组织相应纤维索条区域,RT-qPCR和Western blot检测结果显示肝纤维化组较正常对照组S100A6 mRNA和蛋白表达显著升高(P<0.05)。体外培养原代HSCs过程中,HSCs可自然活化,且随HSCs活化时间延长,S100A6 mRNA和蛋白的表达逐渐增加(P<0.05或P<0.001);干扰S100A6合成后S100A6、Ⅰ型胶原、α-SMA mRNA和蛋白表达均受到不同程度抑制(P<0.05或P<0.001)。结论S100A6在肝纤维化组织和活化HSCs中特异性表达,是一个重要的调控HSCs活化的蛋白,并在肝纤维化的形成机制中发挥一定的作用。

Objective To determine the expression of S100A6 in liver tissues and investigate its role in the activation of hepatic stellate cells(HSCs).Methods Twelve C57BL/6 mice were randomly assigned to control group and liver fibrosis group.The mice in the latter group was administrated with carbon tetrachloride for 6 weeks to induce liver fibrosis.The liver tissue was harvested for Masson staining and S100A6 detection by immunohistochemistry.The expression of S100A6 at mRNA and protein levels was detected by RT-qPCR and Western blotting.After primary HSCs were isolated and cultured,their morphological changes in quiescence and activation were observed,and the mRNA and protein levels of S100A6 were detected after cultured for different time periods.Additionally,siRNA was utilized to interfere S100A6 expression in HSCs,and the mRNA and protein levels of S100A6,CollagenⅠandα-SMA were tested by RT-qPCR and Western blotting.Results Masson staining displayed fibrosis in liver tissue in the mice of the liver fibrosis group,and S100A6 was also significantly expressed in the corresponding fibrous cord area of liver tissue in liver fibrosis group,and its mRNA and protein levels were obviously enhanced in the liver tissue when compared to the control group(P<0.05).In vitro,HSCs auto-activated with the process of culture,and the mRNA and protein expression levels of S100A6 were gradually increased in a time depended manner in the HSCs(P<0.05 or P<0.001).Undoubtedly,the interference of S100A6 by siRNA efficiency dramatically inhibited the mRNA and protein expression of S100A6,CollagenⅠ,α-SMA(P<0.05 or P<0.001).Conclusion S100A6 is specifically expressed in hepatic fibrosis and activated HSCs,and is an important protein regulating the activation of HSCs.Thus,S100A6 might play important role in the pathogenesis of liver fibrosis.