间充质干细胞在常温机械灌注DCD供肝中的保护作用及机制研究

心脏死亡捐献(DCD)供肝因其长时间热缺血损伤及再灌注损伤,严重影响肝移植患者预后.既往研究显示间充质干细胞(MSC)通过多种机制发挥器官缺血损伤修复作用.体外常温机械灌注技术(NMP)是修复DCD供肝的常用方法,我们推测在灌注体系中引入MSC治疗能进一步改善NMP的疗效.本项目拟在前期猪DCD供肝NMP模型基础上,在体外NMP系统中加入MSC,通过HE染色、电镜、TUNEL、ELISA、生化检测方法观察组织病理改变、肝细胞凋亡及炎症因子产生,验证MSC在NMP中对肝损伤的修复作用;并在体内肝移植模型中证实此作用.另外,在体内和体外实验中,探索MSC分泌的VEGF、HGF或TGF-β调控肝细胞凋亡的机制,及通过GM-CSF调控肝内巨噬细胞HO-1表达来发挥炎症调节的作用机制.本课题旨在阐明在NMP体系中MSC对DCD供肝损伤的修复作用及其机制,为临床上应用MSCs保护器官功能提供理论依据.

Liver from Donation after Cardiac Death (DCD) are subjected to long time of warm ischemia injury and reperfusion injury, which serious cause impact on the prognosis of liver transplantation receipient. Previous studies have shown that mesenchymal stem cells (MSC) through a variety of mechanisms play a role in organ ischemia injury protection and immunohomeostasis. In vitro normal mechanical reperfusion technique (NMP) is a common method to repair DCD liver, but its protective function is still inadequate to reverse liver injury. So can combination of MSC and NMP alleviate liver ischemia reperfusion injury from DCD dornor? Based on the early reseach on the aplication of NMP in DCD pig model, the project intends to further unconver the protective effect of MSC with NMP. Index of liver cell apoptosis, liver function and inflammatory factor production would be observed by HE staining, electron microscope, TUNEL, ELISA and biochemical detection method in DCD pig model treated with NMP and MSC or treated respectively. The protective role of MSC and NMP would be furthur confirmed in pig liver transplantation model in vivo. In addition, using in vivo and in vitro experiments, we will identify the mechanism of MSC inhibited hepatocyte apoptosis via secretion of in VEGF, HGF or TGF-beta, and the mechanism of MSC stimulated HO-1 expression in macrophage regulated by GM-CSF. This project will elucidate the protective effect of MSC on the liver injury repair in DCD model, which may provide a theoretical basis for clinical application of MSCs on organ function protection in DCD.