【摘要】 目的观察人肝内胆管上皮细胞(human intrahepatic biliary epithelial cells,HIBEpiC)上皮-间叶转化(epithelial-mesenchymal transitions,EMT)现象,探讨EMT在胆管周围纤维化中的作用及其可能的分子机制。方法HIBEpiC用脂多糖(lipopolysaccharide,LPS)处理48、72 h后,用PCR及Western blot技术检测E-cadherin、S100A4和α-SMA的表达,以及与EMT信号通路相关的TGF-β1的表达;同时用紫杉醇、siRNA smad2/3阻断TGF-β1的作用,探讨TGF-β1/smad2/3信号通路在HIBEpiC发生EMT时的可能作用。结果HIBEpiC经LPS处理后,TGF-β1 mRNA表达于48 h达到高峰,72 h后开始下降,但维持较高水平(P<0.01,P<0.05);EMT标志物E-cadherin mRNA和蛋白表达随培养时间的延长逐渐降低(P<0.01),而S100A4、α-SMA mRNA及蛋白表达明显上调(P<0.01,P<0.05)。紫杉醇可使HIBEpiC中LPS所诱导的TGF-β1、Smad2/Smad3 mRNA表达明显下降,特别是在48 h最明显(P<0.01)。用siRNA技术可使Smad2/3 mRNA及蛋白表达明显下调(P<0.01),同时可使与HIBEpiC共培养EMT标志物E-cadherin mRNA及蛋白质表达上调(P<0.01,P<0.05),S100A4和α-SMA表达明显下调(P<0.01)。结论LPS可诱导HIBEpiC中TGF-β1的表达,使胆管上皮细胞发生EMT,抑制TGF-β1或Smad2/3,可使EMT发生逆转,提示TGF-β1或Smad2/3可能成为预防胆道周围纤维化的一个潜在靶点。更多还原

【Abstract】 Objective To observe the epithelial-mesenchymal transition(EMT)of human intrahepatic biliary epithelial cells(HIBEpiC) and to study its role in bile duct fibrosis and its molecular mechanism.Methods Expressions of E-cadherin,S100A4,α-SMA,and EMT signal-related TGF-β1 in HIBEpiC were detected by RT-PCR and Western blotting 48 and 72 h after treatment of HIBEpiC with lipopolysaccharide(LPS).The effect of TGF-β1 was blocked with paclitaxel and siRNA smad2/3.The role of TGF-β1/ smad2/3 signal pathway in the occurrence of EMT in HIBEpiC was studied.Results The expression of TGF-β1 mRNA reached its peak and began to decrease 48 and 72 h,respectively,after treatment of HIBEpiC with LPS,and still maintained a rather high level(P<0.01,0.05).The expression level of EMT marker,E-cadherin mRNA and TGF-β1 decreased with the prolongation of culture time(P<0.01) while the expression of S100A4,α-SMA mRNA and TGF-β1 significantly increased(P<0.01,0.05),suggesting that paclitaxel can down-regulate the LPS-induced expression of TGF-β1,Smad2 and Smad3 mRNA.siRNA could also down-regulate the LPS-induced expression of Smad2/3 mRNA,S100A4,α-SMA and TGF-β1 while up-regulate the expression of E-cadherin and TGF-β1 in HIBEpiC(P<0.01),indicating that EMT can be reversed.Conclusion LPS can induce expression of TGF-β1 in HIBEpiC,resulting in EMT in biliary epithelial cells,and reverse EMT by inhibiting TGF-β1 or Smad2/3,which may be a potential target for the prevention and treatment of biliary fibrosis.更多还原