肝细胞生长因子调控上皮细胞-间充质细胞转换改善胆管纤维化的机制研究

Regulatory effects of hepatocyte growth factor on epithelium-mesenchymal-transition in improving biliary fibrosis

目的以上皮细胞-间充质细胞转换(epithelial-mesenchymal transition,EMT)在胆道闭锁(biliary atresia,BA)肝外胆管纤维化中的作用及其调节机制为切入点,探讨转化生长因子-β1(transforming growth factor-β1,TGF-β1)对肝外胆管EMT的诱导作用,以及肝细胞生长因子(hepatocyte growth factor,HGF)通过调控EMT来改善肝外胆管纤维化的作用机制。方法提取和培养BALb/c小鼠肝外胆管肝外胆管上皮细胞,按照研究目的不同,将第3次传代后的肝外胆管上皮细胞分为不添加生长因子的正常对照组、添加TGF-β1诱导EMT的TGF-β1组、添加HGF+TGF-β1的HGF干预组、仅添加HGF的HGF对照组和添加TGF-β1+HGF+SU11274的HGF抑制组。用蛋白免疫印记实验检测各组上皮细胞标志物CK19、E-cad和间质细胞标志物α-SMA和S100A4的表达情况。结果蛋白印迹实验显示,对照组CK19、E-cad、α-SMA和S100A4的表达量分别为1.31±0.02、0.90±0.05、0.70±0.07和0.81±0.04。与对照组比较,TGF-β1组上皮细胞标志物CK19(0.75±0.05)和E-cad(0.66±0.06)蛋白表达水平下降,同时间质细胞标志物α-SMA(1.47±0.05)和S100A4(1.49±0.06)蛋白表达水平上升,组间比较,各项细胞因子间差异均有统计学意义(P<0.05)。与TGF-β1组比较,HGF干预组CK19(1.20±0.05)和E-cad(1.12±0.06)蛋白表达水平上升,同时α-SMA(0.71±0.04)和S100A4(0.82±0.07)蛋白表达水平下降,组间比较,各项细胞因子间差异均有统计学意义(P<0.05)。与HGF干预组比较,应用HGF抑制剂SU11274后,HGF抑制组CK1(0.81±0.05)和E-cad(0.99±0.06)蛋白表达水平下降,同时α-SMA(1.25±0.05)和S100A4(1.03±0.07)蛋白表达水平上升,组间比较,各项细胞因子间,除E-cad外,差异均有统计学意义(P<0.05)。结论TGF-β1可诱导肝外胆管上皮细胞发生EMT,HGF可通过调控TGF-β1诱导的EMT来改善肝外胆管纤维化。

ObjectiveThis experiment starts with the role of Epithelial-mesenchymal transition(EMT)in BA extrahepatic bile duct fibrosis and its regulatory mechanism, to investigate the induction of TGF-β1 on extrahepatic bile duct EMT and the mechanism of HGF in improving extrahepatic bile duct fibrosis by regulating EMT.MethodsExtrahepatic bile duct was isolated from BALb/c mice and extraction of extrahepatic bile duct epithelial cells was performed. Immunofluorescence was used to identified the surface markers of extrahepatic bile duct epithelial cells. The proliferation of extrahepatic bile duct epithelial cells was observed after culture.P3 generation extrahepatic bile duct epithelial cells were used for experimental grouping, normal control group(control group), induced EMT group(TGF-β1 group), HGF intervention group (HGF+ TGF-β1 group), HGF control group (HGF group) and HGF inhibition group (TGF-β1+ HGF+ SU11274 group) for a total of 5 groups, with the same incubation time of each factor for 48h. Finally the obtained cells were used to determine the expression of fibrosis related proteins and genes (CK19、E-cad、α-SMA、S100A4) by western blotting. The data were analyzed by Statistics.ResultsWestern blot experiment showed that: Compared with the control group , TGF-β1 could induce down-regulation of epithelial cell markers CK19 (0.75±0.05) and E-cad (0.66±0.06) in extrahepatic bile duct epithelial cells, while up-regulation of mesenchymal cell markers α-SMA (1.47±0.05) and S100A4 (1.49±0.06), the differences between the groups were statistically significant (P<0.05);Compared with TGF-β1 group, the expressions of CK19 (1.20±0.05) and E-cad (1.12±0.06)were up-regulated and α-SMA(0.71±0.04) and S100A4 (0.82±0.07) were down-regulated after HGF intervention, the differences between the groups were statistically significant (P<0.05);Compared with HGF intervention group, after HGF inhibitor SU11274 was applied, CK19 (0.81±0.05) and E-cad (0.99±0.06) expressions were down-regulated, α-SMA (1.25±0.05) and S100A4 (1.03±0.07) expressions were up-regulated, with the exception of E-cad, and the differences between the groups were statistically(P<0.05).ConclusionsTGF-β1 can induce epithelial-mesenchymal transformation of extrahepatic bile duct epithelial cells. HGF can improve extrahepatic bile duct fibrosis by regulating TGF-β1 induced EMT.