肝内胆管上皮细胞上皮-间叶转化的实验研究

Epithelial-mesenchymal transition of intrahepatic biliary epithelial cells

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。

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-eadherin, 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-cadhefin 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-cadhefin 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 tarzet for the prevention and treatment of biliarv fibrosis.