凝血酶-PAR1信号介导脑死亡供肝移植术后缺血再灌注损伤的分子机制研究

Brain death has aggravated the ischemia reperfusion injury in liver transplantation with graft donation after brain death, and its mechanism has not been fully clarified. Thrombin can activate endothelial cells to promote the inflammatory reaction through its receptor (PAR1). CARMA3, Bcl10 and MALT1 combined together, transduct proinflammatory signal from G protein coupled membrane receptor to NF-kappa B. Our previous study found that thrombin activation after brain death, antagonizing PAR1 could inhibit carma3, ICAM-1 and MCP-1 protein expression and p65 nuclear translocation in the liver sinusoidal endothelial cells of brian death rats, and reduce the infiltration of inflammatory cells in the liver tissue. We speculated that the signal pathway of thrombin-PAR1 in the liver sinusoidal endothelial cell could activate the NFkappa B signal to promote ischemia reperfusion injury in liver transplantation with graft donation after brain death through CARMA3 signal transduction. For further confirmation, this paper intends to through gene knock-out, RNA interference and immune coprecipitation, laser confocal and technology, observe thrombin and PAR-1 and inflammatory cells and molecules, histopathologic changes,.ALT and AST etc in vitro and in vivo, and explore the signaling pathway for ischemia reperfusion injury of liver transplantation with graft donation after brain death, in order to provide new thoughts and methods of prevention and treatment for improving the liver quality of brain death donor and reducing ischemia reperfusion injury of liver transplantation with graft donation after brain death.

脑死亡供肝肝移植缺血再灌注损伤（IRI）的具体机制未明。凝血酶能通过其受体(PAR1)激活内皮细胞的促炎反应。CARMA3-Bcl10-MALT1信号转导体能介导G蛋白偶联膜受体激活NF-κB促炎信号。我们前期研究发现大鼠脑死亡后凝血酶激活，拮抗PAR1可以抑制脑死亡大鼠肝窦内皮细胞中CARMA3、ICAM-1和MCP-1蛋白表达上调及p65核转位，并减轻肝组织中炎性细胞浸润。据此推测脑死亡供肝中存在凝血酶通过PAR1调控肝窦内皮细胞CARMA3信号转导体激活NF-κB信号，促进移植术后IRI的发生。为此，本课题拟通过基因敲除、RNA干扰、免疫共沉淀、激光共聚焦等技术，从体内体外层面观测凝血酶、PAR1、炎性细胞及相关分子、组织病理、肝酶学指标等变化，探索该信号通路在脑死亡供肝移植术后IRI的作用，为提高脑死亡供肝质量并减轻移植术后IRI提供新的思路和可能的防治方法。

肝脏移植是治疗终末期肝病的有效方法，而移植肝脏主要来源于脑死亡供体的捐献。研究发现脑死亡状态促进了肝脏炎症反应，加重了移植缺血再灌注损伤，严重影响脑死亡供肝肝移植的效果。因此，研究脑死亡供肝肝移植中缺血再灌注损伤的发生发展机制对减轻肝移植术后并发症、提高肝移植成功率具有重要的临床意义。.在前期研究中我们发现凝血酶片段、PAR1在大鼠脑死亡后显著升高，炎症细胞、肝窦内皮细胞被激活。通过构建PAR1敲除大鼠模型、进行体内外实验，我们发现凝血酶通过激活PAR1-CARMA3信号转导体炎性活化肝窦内皮细胞加重肝脏缺血再灌注损伤。.同时，我们还发现Steap3、TRIM27在脑死亡供肝肝移植术后变化显著，在肝脏缺血再灌注损伤的炎症反应和细胞凋亡中发挥重要作用。在分子机制方面，我们证实Steap3通过调控 NF-κB 和 MAPK信号通路，TRIM27通过调控TAK1-JNK/p38 信号通路介导肝脏缺血再灌注损伤。此外，我们还证实针对Steap3和Trim27进行靶向治疗潜在的临床意义。.最后，我们从圣约翰草、决明子种子、银杏叶中分离、筛选到具有抗凝血酶作用的金丝桃素、橙黄决明素，美决明素和部分双黄酮成分（银杏双黄酮，异银杏双黄酮，白果双黄酮，穗花杉双黄酮），通过分子对接证实其潜在机制。.总之，我们阐明了凝血酶、Trim27、Steap3在脑死亡供肝肝移植后缺血再灌注损伤的作用、分子机制以及临床应用前景，为肝脏缺血再灌注损伤的精准治疗提供靶点。此外，我们的研究还为开发新型改善缺血再灌注损伤药物、抗凝血酶药物提供合理依据。