【作者】 黄海涛；

【导师】 凌琪；

【作者基本信息】 浙江大学 ， 外科学（专业学位）， 2020， 硕士

【摘要】 背景:近年来肝移植已被认为是一种治疗各种终末期肝病的有效治疗手段。移植肝作为受体新的免疫代谢器官,在植入受体后立即发生移植肝内的免疫重塑。其中受体来源的免疫细胞(Recipient-derived immune cells,RICs)被大量招募和激活,并与移植肝内供体来源免疫细胞(Donor-derived immune cells,DICs)以及肝细胞发生交互,对受体的新陈代谢和免疫功能产生重要影响,甚至促进肝移植术后多种并发症包括代谢紊乱,肿瘤复发等发生发展。因此,对肝移植术后移植肝免疫重塑的深入研究有助于进一步理解肝移植术后并发症的发病机制并制定治疗策略。目的:探索小鼠肝移植术后,移植肝内免疫重塑的过程。方法:利用全身表达eGFP荧光蛋白的雌性转基因小鼠(C57BL/6J背景)作为供体,野生型雄性C57BL/6J小鼠作为受体构建小鼠原位肝移植模型。应用单细胞测序、质谱流式技术,免疫组化等方法探究在小鼠肝移植术前及术后不同时间点移植肝内免疫重塑的情况。结果:肝移植术后12小时,肝内供体来源细胞急剧下降至34%,而受体来源细胞的占比从0%急剧增加至66%。其中绝大多数细胞亚群以受体来源为主,但也有部分亚群仍以供体细胞为主,包括肝细胞,内皮细胞,以及部分自然杀伤细胞(Natural killer cells,NK)和M2型巨噬细胞。进一步探索肝移植术后两周内肝内免疫重塑情况发现,DICs在两周后仅占总体免疫细胞5.6%,而RICs的占比已达94.4%。就细胞亚群而言,仍有部分M2型巨噬细胞和NK细胞亚群仍保留一半左右DICs。鉴于巨噬细胞在肝脏稳态中的重要作用,对不同来源的巨噬细胞功能研究发现,受体来源巨噬细胞中(Recipient-derived macrophages,RMs)促炎的M1型巨噬细胞占据重要比例,而在供体来源巨噬细胞(Donor-derived macrophages,DMs)中促炎的M1及抗炎的M2型巨噬细胞均占据重要比例。结论:该项研究首次探索了小鼠肝移植术后肝内免疫重塑过程。RICs在移植术后将快速占据移植肝(>90%),但是DICs在部分M2型巨噬细胞及NK细胞中仍占据重要位置并发挥着作用。在特定的环境信号下,由于巨噬细胞及NK细胞的异质性,供体和受体之间的遗传学差异可能会对巨噬细胞及NK细胞产生重大影响。针对不同来源的巨噬细胞及NK细胞的深入研究可能会有助于加深移植肝内免疫重塑及部分移植后并发症发病机制的理解。更多还原

【Abstract】 Background:In recent years,liver transplantation provides a cure for various end-stage liver diseases.After transplanted into the recipient,the liver graft becomes the recipient’s new immune metabolic center and immediately undergoes immune remodeling by interacting with the recipient.Countless recipient-derived immune cells(RICs)were activated and recruited into the liver graft.The interaction between the RICs and donor-derived cells(e.g.donor-derived immune cells,DICs or hepatocytes)affects immunological function and metabolic homeostasis,which even promotes a variety of complications,including metabolic disorders and tumor recurrence.Therefore,the immune remodeling in the graft liver is deserved for further exploration to understand the pathogenesis of post-transplanted complications and develop treatment strategiesAims:To explore the immune remodeling in the liver graft following liver transplantation.Methods:We explore the immune remodeling in the liver graft during the perioperative period in a murine orthotopic liver transplantation model.Female mice liver expressing systemical eGFP were transplanted to wild type male recipients.Single cell RNA sequencing(scRNA-seq),mass spectrometry(CyTOF),immunohistochemistry and flow cytometer were used to monitor the immune remodeling in the liver graft.Results:During the first 12h,donor-derived cells in the liver graft sharply decreased to 34%,while the proportion of recipient-derived cells increased sharply from 0%to 66%.Recipient-derived cells became predominant in most of the cell subgroups,but others were still dominated by donor-derived cells,including hepatocytes,endothelial cells,and some natural killer cells(NK)and M2 macrophages.Two weeks later,we found that DICs only accounted for 5.6%in total immune cells,while 94.4%for RICs.Upon a closer evaluation of the subtypes of immune cells,some M2 macrophages and NK cell subsets still retain about half of DICs.Considering the important role of macrophages in liver homeostasis,we investigated the function of macrophages from donor and recipient.We found that pro-inflammatory M1 macrophages in charge of the recipient-derived macrophages(RMs),while both pro-inflammatory M1 and anti-inflammatory M2 macrophages contributed to the donor-derived macrophages(DMs).Conclusions:In conclusion,this study is the first to explore process of immune remodeling in the liver graft.It was striking that RICs took over the liver graft(>90%)so quickly,but RICs still played a role in M2 macrophages and NK cells.Because of the heterogeneity of macrophages and NK cells,differing genetics between donor and recipient may have a considerable impact on the function of macrophages and NK cells in response to specific environmental signals.Intensive research on macrophages and NK cells from donor or recipient may help to deepen the understanding of immune remodeling in liver graft and the pathogenesis of post-transplant complications.更多还原