5-氟尿嘧啶抑制TGF-β1诱导人肝内胆管上皮细胞间质化

Inhibitory effect of 5-FU on TGF-β1-induced human intrahepatic biliary epithelial-mesenchymal transition

目的探讨5-氟尿嘧啶对TGF-β1诱导人肝内胆管上皮细胞间质化的抑制作用,并探讨其作用机制。方法原代培养人肝内胆管上皮细胞,角蛋白19荧光染色鉴定;细胞分为:对照(normal)组,TGF-β1(TGF-β1)组,5-氟尿嘧啶(TGF-β1+5-FU)组;免疫荧光染色观察CK-19、E-cadherin、vimentin和α-SMA标记蛋白;Western blot检测细胞标记蛋白及TGF-β1表达量;Real-time PCR检测细胞Ⅰ、Ⅲ型胶原及TGF-β1 mRNA含量。结果原代培养的人肝内胆管上皮细胞呈多边形或者锥形,CK-19荧光染色为胞质着色;TGF-β1诱导72 h后,胆管上皮出现间质化表现,与正常组比较,vimentin、α-SMA和TGF-β1表达明显增强(P<0.05),CK-19和E-cadherin表达显著减弱(P<0.05),Ⅰ、Ⅲ型胶原及TGF-β1 mRNA含量显著升高(P<0.05);5-FU抑制胆管上皮间质化,与TGF-β1组比较,CK-19和E-cadherin表达增强(P<0.05),vimentin、α-SMA和TGF-β1表达明显减弱(P<0.05),TGF-β1 mRNA和Ⅲ型胶原mRNA含量明显降低(P<0.05)。结论 5-FU通过下调TGF-β1的表达抑制胆管上皮细胞间质化。

Objective To investigate the inhibitory effect of 5-FU on human intrahepatic biliary epithelial cells with TGF-β1-induced mesenchymal transition and potential mechanism. Methods The primary epithelial cells from hu- man intrahepatic bile ducts were cultured, the cells were identified by immunofluorescence microscopy. The cells were randomly divided into 3 groups： normal control group, TGF-β1 group and TGF-β1＋5-FU group. The expres- sion of CK- 19, E-cadherin, vimentin and α-SMA protein were measured by immunofluorescence microscopy and Western blot assay. Western blot was used to detect the expression levels of TGF-β1. Real-time PCR to detect mR- NA expression of TGF-β1, Ⅰ and collagen typeⅢ. Results The primary epithelial cells of intrahepatic bile ducts were successfully cultured. In TGF-β1 group, the protein expression of vimentin, α-SMA and the mRNA expression levels of I and collagen type Ⅱ were significantly higher than that of normal control group （P〈0.05） , the protein expression of CK-19 and E-cadherin protein were lower than that of normal control group（P〈0.05） ,TGF-β1protein and mRNA expressions were significantly higher compared with that of normal control group（ P〈0. 05）. In TGF-β1＋5-FU group, the protein expression levels of CK-19 and E-cadherin protein were higher than that in normal control group （P〈0. 05）, the protein expression level of vimentin, α-SMA and the mRNA expression level of collagen type Ⅲ were significantly lower than that of TGF-β1 group（ P〈0. 05）, while there was no significantly difference in collagen typeⅠmRNA between the two groups（P〉0. 05）, TGF-β1 protein and mRNA expressions were significantly lower as compared with that of normal TGF-β1 group（P〈0. 05）. Conclusions 5-FU could inhibit TGF-β1-induced biliary epithelial-mesenchymal transition with down-regulated expression of TGF-β1.