JunD通过NF-кB通路活化Snail促进EMT形成及其在脑死亡供肝移植术后缺血性胆道损伤中的作用

Ischemic-type biliary injury (ITBI) is a common complication after liver transplantation with graft donation after brain death. The underlying mechanism of ITBI remains to be determined. It was reported that epithelial-mesenchymal transition (EMT) of intrahepatic biliary epithelial cells is a key molecular basis for the development of ITBI. In preliminary studies with microarray assay of gene expression and rat models of liver transplantation with graft donation after brain death, we found that the expression of JunD, Snail and fibroblast-specific protein 1 (FSP-1), was highly upregulated, and NF-кB signaling was activated in intrahepatic biliary epithelial cells. These evidences led us to hypothesize that EMT mediated by a signaling pathway consisting of JunD ,NF-кB and Snail may involve in regulation of the development of ITBI. In this proposal, we seek to further investigate JunD-NF-кB-Snail-EMT signaling pathway. In collaboration with monitoring of serum biomarkers of biliary injury and observation of morphological alternation of intrahepatic biliary epithelial cells, we will elucidate the role and molecular mechanism of this pathway in regulation of ITBI development following liver transplantation with graft donation after brain death. We will employ complementary approaches that include gene transfection, small hairpin RNA, co-immunoprecipitation, confocal microscopy, etc., combined with primary culture of biliary epithelial cells, rat models of liver transplantation and clinical liver transplantation analysis in our study. This study will provide theoretical basis to explore mechanism-driven approaches for future prevention and treatment of ITBI following liver transplantation.

缺血性胆道损伤（ITBI）是脑死亡供肝移植后的常见并发症，详细机制尚待阐明。研究报道ITBI的发生可能与胆道上皮细胞发生上皮间质转化（EMT）有关。我们前期利用基因芯片发现脑死亡大鼠肝脏中JunD持续活化，进一步研究发现脑死亡大鼠供肝移植后胆道上皮细胞中JunD上调、NF-кB活化、Snail及间质细胞标志物(FSP-1)表达升高。据此推测可能存在JunD—NF-кB—Snail—EMT信号通路介导脑死亡供肝移植后ITBI的发生。本项目拟采用基因转染、shRNA干扰、免疫共沉淀、激光共聚焦等技术从原代胆管上皮细胞、大鼠肝移植模型、临床肝移植标本等层面，检测并研究JunD、NF-кB、Snail、FSP-1等相关分子信号的改变及其与胆道损伤血清学、形态学指标变化的关系，探索该信号通路参与脑死亡供肝移植后ITBI发生的机制，为减轻脑死亡供肝移植后的缺血性胆道损伤提供理论基础和可能的防治方法。

项目背景：肝脏移植是治疗织末期肝病的有效方法。脑死亡供者已成为国内、外肝脏移植供肝的重要来源。研究报道脑死亡来源的供肝移植术后胆道并发症较高，而缺血性胆道损伤是移植术后胆道并发症的主要原因。缺血再灌注损伤和胆管细胞EMT是脑死亡供肝移植术后缺血型胆道损伤的主要原因。JunD和Steap3分别在缺血型胆道损伤和缺血再灌注损伤后高表达，并且是调控纤维化、炎症和凋亡等生物学过程的关键因子，但JunD和Steap3是否参与脑死亡供肝移植术后缺血型胆道损伤的调控及其相关分子机制尚未阐明。研究内容：本研究通过建立大鼠脑死亡供体肝移植模型、小鼠缺血再灌注损伤模型，结合基因敲除、基因克隆、免疫共沉淀、慢病毒感染、原代肝细胞分离培养等实验技术，从体内及体外水平研究脑死亡供肝移植术后缺血型胆道损伤的机制。研究结果：1、脑死亡状态通过加重肝脏缺血再灌注损伤从而促进脑死亡供肝移植术后缺血型胆道损伤；2、脑死亡供肝移植术后存在大量差异表达基因，调控脑死亡供肝肝移植术后肝损伤和胆道损伤；3、JunD、Steap3高表达是脑死亡供肝移植术后缺血型胆道损伤的基本特征； 4、JunD通过调控下游NFκB通路进而诱导EMT，参与脑死亡供肝移植术后缺血型胆道损伤；5、Steap3通过促进炎症反应和细胞凋亡加重肝脏缺血再灌注损伤；6、Steap3通过激活NFκB和MAPK信号通路调控肝脏缺血再灌注损伤。7、Steap3调控肝脏缺血再灌注损伤依赖于和TAK1互作。研究结论：通过上述研究，本项目阐明了JunD和Steap3对脑死亡供体肝移植术后缺血型胆道损伤调控的分子机制，为脑死亡供体供肝移植术后缺血性胆道损伤的防治提供了新的思路和潜在的治疗靶点，为临床应用提供了理论指导。