trps1调控胆管上皮细胞上皮-间质转化的实验研究 - 第三军医大学学报.doc

Trps1调控胆管上皮细胞上皮-间质转化的实验研究 程 哲，田 驹，郑树国 400038重庆，第三军医大学西南医院全军肝胆外科研究所 [摘要] 目的 探讨毛发鼻指（趾）综合征1型相关基因（tricho-rhino-pharyngeal syndrome type 1，Trps1）在冷保存再灌注损伤（cold preservation reperfusion injury，CPRI）诱导人肝内胆管上皮细胞（human intrahepatic biliary epithelial cells，HIBECs）上皮-间质转化（epithelial-mesenchymal transition，EMT）中的作用。方法 将HIBECs置于4℃ UW液中缺氧保存12、24、48h后，再转移至37℃普通培养基中孵育1h，PCR及Western blot检测上皮标志物E-cadherin、CK19，间质标志物Vimentin、α-SMA及目标基因Trps1的表达。用Trps1腺病毒或Trps1 siRNA载体转染HIBECs后，重复CPRI诱导实验，探讨Trps1对HIBECs EMT的调控作用。结果 体外模拟CPRI处理HIBECs，随着冷保存时间的延长，上皮标志物E-cadherin、CK19 mRNA和蛋白表达持续减少（P=0.018,0.011;P=0.039,0.044），间质标志物Vimentin、α-SMA表达持续增加（P=0.002,0.026;P=0.035,0.016）；冷保存12h，细胞内Trps1 mRNA和蛋白表达增加（P=0.018,0.031），其后随冷保存时间延长持续减少（P=0.026,0.009）。相关分析显示在该过程中，Trps1与上皮标志物表达正相关（r=0.981,0.913;P=0.000,0.002），而与间质标志物表达负相关（r=-0.711,-0.847;P=0.009,0.005）。Trps1腺病毒转染HIBEC后，重复上述CPRI诱导实验，E-cadherin表达增加（P=0.002,0.000），Vimentin表达减少（P=0.000,0.017），CPRI诱导HIBECs EMT效应受到抑制；Trps1 siRNA转染HIBECs后，重复上述CPRI诱导实验，E-cadherin表达减少（P=0.032,0.044），Vimentin表达增加（P=0.047,0.031），加强CPRI诱导HIBECs EMT效应。结论 体外模拟CPRI可诱导HIBECs发生EMT，Trps1参与调控HIBECs EMT，发挥重要的拮抗作用并可能逆转该过程。 [关键词] Trps1；胆管上皮细胞；上皮-间质转化；冷保存再灌注损伤 [中图法分类号] R361+.3; R394.3; R657.4+6 [文献标志码] A Trps1 regulates epithelial-mesenchymal transition of intrahepatic biliary epithelial cells Cheng Zhe, Tian Ju, Zheng Shuguo（Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China, 400038, China） [Abstract] Objective To observe the epithelial-mesenchymal transition (EMT) of human intrahepatic biliary epithelial cells (HIBECs) induced by cold preservation reperfusion injury (CPRI), and to study the role of tricho-rhino-pharyngeal syndrome type 1 (Trps1) in the occurrence of HIBECs’ EMT. Methods First HIBECs were placed in 4℃ UW solution for 12, 24, 48 hours, then were incubated in 37℃ normal serum-containing medium for 1 hour, respectively simulating the cold preservation stage and the reperfusion stage of CPRI