脂多糖对人肝内胆管上皮细胞IL-6/STAT3信号通路的影响

摘要

目的 探讨脂多糖(LPS)对正常人肝内胆管上皮细胞(HiBECs)白介素(IL)-6/STAT3信号通路的影响.方法 HiBECs细胞常规体外培养,使用不同浓度的LPS(0、0.1、1、2、4、8 μg/ml),分别作用该细胞24、48、72 h后,ELISA法检测IL-6的表达水平,确定LPS的最适刺激浓度及最佳刺激时间,再以其进行后续实验;RT-qPCR法测定c-Myc mRNA、Mcl-1 mRNA的表达情况;Western blot法测定p-STAT3/STAT3蛋白比例及c-Myc、Mcl-1蛋白的表达情况.结果 LPS能够促进HiBECs细胞IL-6分泌,并且在4 μg/ml、24 h时IL-6的分泌量达最大(F=16.492、17.763,P<0.001、P<0.01);LPS刺激HiBECs细胞后,c-Myc mRNA、Mcl-1 mRNA表达上调 (P<0.01);p-STAT3/STAT3蛋白比例增高(P<0.05),c-Myc(P<0.001)、Mcl-1(P<0.05)蛋白表达增多.结论 LPS能够激活HiBECs细胞IL-6/STAT3信号通路,并且可能通过该信号通路促进Mcl-1、c-Myc的表达.%Objective To investigate the expression of IL-6/STAT3 signal pathway induced by lipopolysaccharide (LPS) on human intrahepatic biliary epithelial cells (HiBECs) in vitro.Methods The HiBECs were cultured in vitro.The cells were stimulated by LPS at different concentrations (0,0.1,1,2,4,8 μg/ml) and different time (24,48,72 h).The expression of IL-6 was measured by ELISA to determine the best concentration and time of LPS.Then we chose the best concentration and time of LPS to intervene the cells in the further experiments.The levels of mRNA expression of c-Myc and Mcl-1 were detected by fluorescence quantitative reverse transcrption PCR (RT-qRCR).The proportion of p-STAT3/STAT3 protein and the expression of c-Myc and Mcl-1 protein in HiBECs were analyzed by Western blot.Results LPS upregulated the expression of IL-6, and reached its peak at 4 μg/ml and 24 h(F=16.492,17.763,P<0.001,P<0.01).LPS increased the mRNA expression of c-Myc and Mcl-1(P<0.01).Meanwhile, The ratio of p-STAT3/STAT3 (P<0.05) protein and the expression of c-Myc (P<0.001) and Mcl-1 (P<0.05) protein were upregulated after stimulating with LPS.Conclusion The IL-6/STAT3 signal pathway can be activated by LPS in HiBECs, and LPS may promote the expression of c-Myc and Mcl-1 through this signal pathway.