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

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.

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.

移植器官所必须经历的缺血，冷保存，再灌注给移植疗效带来了极大的影响甚至威胁患者生命。. 本团队率先建立无缺血肝移植技术IFLT，实现了移植过程中不中断血流，成功避免了器官缺血带来的损害，并将理念向肾、肺、心等器官延伸，使器官移植进入“热移植”时代。首先，成功研制多器官机械灌注系统，实现保存过程中器官的常温、有氧血供，同时创新供肝获取与植入外科技术，实现供肝获取和植入过程的血流不中断，率先进行大量动物实验，建立稳定的大动物模型，随后成功实施全球首例临床无缺血肝移植、肾移植。而后本团队继续探索无缺血技术避免缺血再灌注损伤的机制，发现了传统缺血再灌注损伤相关的基因和代谢物以及炎症因子等，从多组学验证无缺血器官移植技术优于传统技术。转录组学分析显示常规技术组分别有805个基因表达显著上调和58个基因表达显著下调，而IFLT供肝复流后仅有11个基因表达显著上调和11个基因表达显著下调。通过病理学、RT-PCR、免疫组化和Western blot等方法检测临床供肝标本，发现在IFLT中，未明显出现部分肝细胞凋亡或坏死、炎症因子和趋化因子释放、血管内皮损伤和炎症信号通路激活等病理生理过程。. 除此以外，本团队依托机械灌注建立器官医学平台，将疾病机制的探索放到了器官整体水平，并进行了一系列的靶向机制研究和预后评估。建立首个基于多个神经病学参数预测供肝IRI的nomogram模型，并且应用功能影像学评估供肝的原发疾病，同时筛选出可预测移植肝IRI的分子标记物。