IL-17A诱导肝内胆管上皮细胞上皮-间质转化在PBC胆管纤维化中的意义

Primary biliary cirrhosis (PBC) is an autoimmune cholestatic liver disease characterised by antimitochondrial antibody against the pyruvate dehydrogenase complex and chronic non-suppurative destructive cholangitis, with a apparent injury to intrahepatic biliary epithelial cells (IBEC), finally causing biliary cirrhosis. Some studies have found that the epithelial-mesenchymal transition (EMT) is closely related to the development of liver fibrosis. Inhibiting the EMT can reduce or reverse hepatic fibrosis, even delay the process of cirrhosis of the liver. Therefore EMT has drawn wide attention in the research on liver fibrosis and related drugs in recent years. IL-17A is a newly discovered pro-inflammatory cytokines, which has been recently identified as a key cytokine involved in numerous autoimmune processes. However, its role in PBC remains unclear. Does IL-17A inducing IBEC-EMT contribute to the pathogenesis of biliary fibrosis in PBC? Taken together, It's worthy to focus on the contribution of IL-17A to the IBEC-EMT and the biliary fibrosis and the possible mechanism in PBC. In order to comprehensive understanding this question, this study through the vivo and vitro experiments to observe the role of IL-17A in IBEC-EMT and biliary fibrosis. Firstly, we will detect the pathological changes of liver tissue of PBC patients. Immunohistochemical staining will be used to detect the level of biliary fibrosis, IL-17A, Th17 cells and IBEC-EMT. Secondly, we will investigate the signaling mechanism mediating the regulatory role of IL-17A in IBEC-EMT. Thirdly, using PBC models of the C57BL/6 WT mice, along with IL-17A neutralizing antibody, we further observe the role of the above pathological processes in the mouse model of PBC, and eventually help looking for blocking PBC liver fibrosis development strategies and methods to provide the experimental basis.

原发性胆汁性肝硬化(PBC)最终结局是肝硬化，典型病理学改变是肝内小胆管慢性非化脓性破坏性胆管炎及胆汁性肝硬化，肝内胆管上皮细胞(IBEC)损害明显。上皮-间质转化(EMT)与肝纤维化的发生发展密切相关，通过抑制EMT可减轻或逆转肝纤维化，从而延缓肝硬化进程。体内EMT受多因素影响，IL-17A在EMT中作用近年倍受到重视。因组织器官和疾病的差异，IL-17A在细胞EMT及器官纤维化中的研究结论不一。当前研究尚未明确IL-17A诱导IBEC-EMT及其分子机制，以及该病理过程在PBC中胆管纤维化中的价值。课题组前期研究已证实：PBC患者胆管上皮细胞发生EMT，IL-17A参与此病理过程。本研究拟继续深入探索IL-17A诱导IBEC-EMT分子机制，并在PBC小鼠模型上全面观察IL-17A诱导IBEC-EMT在PBC胆管纤维化中的意义，为寻找阻断PBC肝纤维化发展提供策略和方法提供实验依据。

原发性胆汁性胆管炎（PBC）最终结局是肝硬化，典型病理学改变是肝内小胆管慢性非化脓性破坏性胆管炎及胆汁性肝硬化，肝内胆管上皮细胞（IBEC）损害明显。上皮-间质转化（EMT）与肝纤维化的发生发展密切相关，通过抑制EMT可延缓或阻断肝纤维化进程。当前研究未明确IL-17A是否诱导IBEC-EMT？其分子机制如何？该病理过程在PBC中胆管纤维化中的价值如何？为此，课题组进行了以下几个方面的研究：（1）通过PBC病例临床研究，明确PBC患者是否发生IBEC-EMT，肝内胆管中IL-17A表达变化以及外周血IL-17A等炎性细胞因子的变化。（2）体外培养IBEC，观察IL-17A诱导IBEC-EMT，并探索其可能的分子机制，包括：建立EMT经典细胞模型；观察IL-17A是否可诱导出IBEC-EMT；应用siRNA沉默IBEC相应靶基因，探索IL-17A诱导IBEC-EMT分子机制。（3）IL-17A和TGF-β1共同干扰培养IBEC，观察IL-17A对TGF-β1诱导IBEC-EMT的影响。（4）动物实验验证IL-17A在PBC中IBEC-EMT及其在胆管纤维化中的作用。通过以上一系列的体内外研究，主要研究结果：（1）PBC患者多项生理生化指标异常，外周血清IL-17A等多种炎性细胞因子水平升高。（2）PBC患者肝内胆管上皮细胞上皮标志物E-cadherin明显降低，间质标志物Vimentin明显增高，同时IL-17A、IL-17RA表达升高，提示在PBC发展过程中伴随IBEC-EMT的发生。（3）IL-17A干扰培养IBEC，转染siRNA-Act1可阻断IBEC-EMT过程，同时下游信号通路上Act1、P65/p-P65、p-IκB等多种信号蛋白表达异常。（4）IL-17A和TGF-β1共同干扰培养诱导IBEC-EMT较单独应用明显。（5）PBC小鼠模型体内实验表明：IL-17A体内可诱导IBEC-EMT，参与PBC纤维化过程，IL-17A中和抗体可减缓这一病理过程。该研究结果表明：IL-17A参与PBC肝内胆管IBEC-EMT病理过程，可能通过IL-17A－IL-17RA－Act1－NF-κB信号通路发挥作用，IL-17A中和抗体可减缓PBC病理过程。该研究最终为寻找阻断或延缓PBC肝纤维化进程的策略和方法提供理论及实验依据。