Trps1调控胆管上皮细胞转分化在DCD肝移植术后胆管纤维化中的作用及机制

Ischemic-type biliary lesions(ITBL) is a common disease following liver transplantation (LT) with the donation after cardiac death (DCD) ,the bile duct fibrosis is the important pathogenesis of ITBL,and the epithelial-mesenchymal transition (EMT) of bravery epithelial cells (BEC) is the important mechanism of biliary duct fibrosis. Trps1 is an important regulator of transdifferentiation from embryonic mesenchymal cells to the epithelial cells, which plays a key role in negative regulation of EMTs in a variety of cells. We observed that Trps1 inhibited the biliary fibrosis in the liver allograft by negatively regulating the EMT of BECs in our previous study.Based on our results and Ischemic-type biliary lesions(ITBL) is a common complication following liver transplantation (LT) with the donation after cardiac death (DCD) , which has an adverse effect on long-term survival of recipients .The biliary fibrosis is the pathological basis of ITBL ,and the epithelial-mesenchymal transition (EMT) of biliary epithelial cells (BECs) is the pathogenesis of biliary duct fibrosis. Trps1 is an important regulator of transdifferentiation in embryo, which plays a key role in negative regulation of EMT in a variety of cells. We observed that Trps1 inhibited the biliary fibrosis in the liver transplantation with the DCD by negatively regulating the EMT of BECs in our previous study .Based on our results and rencent literatures, we hypothesized that Trps1 may inhibit or reverse the biliary fibrosis of liver transplantation through directly binding to STAT3 and negatively regulating the EMT of BECs. The presernt study aims to validate our hypothesis on the cultured BECs and DCD models of rats and gene knockout mouse. Multiple methods including immunohistochemistry, RT-PCR, Western blotting and CHIP, luciferase report, laser confocal are applied to observe the effects of Trps1 on morphological changes, epithelial and mesenchymal markers expression and STAT3 nuclear translocation of BECs. By up-regulating, silenceing or knocking down of Trps1 gene, we had the opportunity to clarify its effects and pathogenesis of BECs’ transdifferentiation in the biliary fibrosis of liver transplantation with the DCD.

缺血型胆道病变（ITBL）严重影响心脏死亡捐献（DCD）肝移植受者长期生存，其病理基础是胆管纤维化，胆管上皮细胞(BEC)上皮间质转分化(EMT)是其重要发病机制。作为胚胎期间质向上皮转分化的重要调控因子，Trps1对EMT具有关键负向调控作用。课题组在预实验中发现Trps1参与了DCD肝移植术后胆管纤维化的负向调控过程。结合文献，我们推测：Trps1可能通过直接结合STAT3发挥对BEC EMT的负向调控作用，进而抑制或逆转移植肝胆管纤维化。本课题拟在体外培养的BEC以及大鼠、基因敲除小鼠DCD肝移植模型上，采用免疫组化、RT-PCR、Western blotting、CHIP、荧光素酶报告及激光共聚焦等方法深入研究BEC形态学、上皮、间质标志物、STAT3表达及其核转位改变，并通过上调、沉默或敲除Trps1，阐明其调控BEC转分化在DCD肝移植术后胆管纤维化中的作用及可能机制。

缺血型胆道病变（ITBL）严重影响心脏死亡捐献（DCD）肝移植受者长期生存，其病理基础是移植肝胆管上皮细胞缺血再灌注损伤（IRI）后异常修复所致的胆管纤维化，胆管上皮细胞(BEC)上皮间质转分化(EMT)是其重要发病机制。Trps1是胚胎期间充质细胞向上皮细胞转分化的重要调控因子，对多种细胞EMT起关键的负向调控作用。但Trps1是否参与了DCD移植肝胆管IRI后修复过程，并通过对BEC EMT的关键负向调控进而抑制移植肝胆管纤维化，目前尚未见报道。为此，本项目设计了三部分研究内容：①临床病例资料分析，收集DCD肝移植术后因缺血型胆道病变行二次肝移植患者的病肝标本，检测Trps1、EMT相关标志物的表达及胆管周围胶原含量变化，并对IL-6、STAT3及p-STAT3进行检测，发现Trps1 参与了DCD肝移植术后胆管纤维化的发病过程并与BEC EMT和移植肝胆管纤维化呈负相关，其作用与IL-6/STAT3 信号通路相关。 ②细胞实验，以体外培养的人肝内胆管上皮细胞（HiBEC）为研究对象，成功建立了DCD细胞模型，深入探讨了Trps1对BEC EMT的调控作用及分子机制，发现STAT3是Trps1抑制BEC EMT的关键下游分子及Trps1通过IL-6/STAT3信号通路调控胆管上皮细胞转分化的关键作用。③动物实验：在重建肝动脉血供的大鼠DCD 肝移植模型上，检测了移植肝胆管上皮细胞EMT相关标志物表达及胶原含量变化，分别用含Trps1基因全长的慢病毒和Trps1 shRNA慢病毒转染BEC在体上调和下调 Trps1的表达，并给予STAT3阻断剂Rapamycin，在体条件下证实了Trps1 对DCD移植肝胆管纤维化具有关键负向调控作用及机制。.本项目首次提出并验证了Trps1参与了DCD肝移植术后胆管纤维化过程，并通过对BEC EMT的关键负向调控作用，进而抑制或逆转抑制胆管纤维化进程的研究假说，拓展了移植肝胆管纤维化发病机制的理论，为临床DCD肝移植术后缺血型胆道病变的防治提供了新的策略。本项目研究结果也将为临床常见的损伤性胆管狭窄、肝胆管结石病等相关疾病的理论研究和临床诊治提供新的线索。.本项目研究工作已发表SCI论文2篇，在投SCI论文 1篇、中文论文1篇。课题组在本项目研究基础上滚动申请并获批了国家自然科学基金面上项目1项（编号：81972303）。