间质干细胞通过调节性B细胞治疗肾移植慢性排斥的机制研究

Chronic rejection is one tough barrier to further improve long-term graft survival in kidney transplantation. There are unfortunately no effective regimens to overcome it. In the completed clinical trial, we have testified the efficacy and safety of clinical application of mesenchymal stem cells (MSCs) in kidney transplantation to prevent acute rejection, and the results have been published on Transplantation, 2013, 95:161. In the subsequent experiments, we found MSCs transfusion attenuated chronic rejection and significantly improved graft function in a rat model of allogeneic kidney transplantation. In the same study, we also observed a significant increase of peripheral CD5+ B cells in spleens of recipient rats after MSCs treatment. Results of following primary experiments demonstrated that MSCs could directly increase CD5+ B cells counts in vitro, and most importantly, enhance the inhibitory function of CD5+ regulatory B cells (Bregs). In this study, we are going to introduce CD19 knockout mice to determine the key role of CD5 regulatory B cells in MSC treatment of renal allograft chronic rejection, and investigate the effect of CD5 Bregs on the phenotype and function of effector immune cells in the chronic rejection model. Furthermore, we will explore the mechanisms of MSCs on CD5 Bregs count and function, including the effect of MSCs on CD5 Bregs apoptosis and proliferation, and MSCs-secreting soluble cytokines, such as IDO and PEG2. Our research may provide new insights into the mechanism of MSCs therapeutic function and reveal a novel regulation mechanism of chronic rejection in allogeneic kidney transplantation.

慢性排斥是影响肾移植远期疗效的重要障碍，尚无有效治疗手段。前期临床试验证实了间质干细胞（MSCs）预防肾移植急性排斥的安全性和有效性（Transplantation. 2013,95：161）。进一步利用大鼠肾移植模型发现，MSCs能减轻慢性排斥，改善移植肾功能，且受体脾脏CD5+B细胞明显增加。体外预实验表明，MSCs能直接增加CD5+调节性B细胞（Bregs）数量，并增强其免疫抑制功能。据此，本项目拟采用CD19基因敲除小鼠证实CD5+Bregs在MSCs治疗慢性排斥中的关键作用；过继输注CD5+Bregs探讨其在动物体内对慢性排斥相关免疫效应细胞的影响；并通过体外实验明确MSCs影响CD5+Bregs数量和功能的机制，包括MSCs对其细胞凋亡和增殖的影响，及MSCs分泌IDO、PGE2等关键因子的作用。本项目可为MSCs治疗肾移植慢性排斥的作用机制提供新的科学解释和实验依据。

慢性排斥是影响肾移植远期疗效的重要障碍，尚无有效治疗手段。本课题明确了间充质干细胞对大鼠肾移植慢性排斥的治疗作用，发现MSCs治疗后CD5+调节性 B 细胞比例明显增高，血清IL-10水平增高；进一步采用anti-CD20 mAb清除外周成熟B细胞，MSCs治疗作用消失；静脉输注CD5+CD19+B细胞后，MSCs重新起到治疗慢性排斥的作用，说明CD5+CD19+B细胞在MSCs治疗慢性排斥中发挥重要作用。进一步研究发现，MSCs共培养能够促进CD5+ B细胞增殖，抑制CD5+ B细胞凋亡，是通过分泌可溶性因子发挥作用，IDO可能是参与其中的重要作用蛋白。静脉输注CD5+CD19+B细胞，受体淋巴结和脾脏内CD4+CD25+Foxp3+调节性T比例升高，外周血清IL-10水平明显升高，提示CD5+B细胞可能通过分泌IL-10诱导Tregs发挥免疫抑制作用。研究过程中发现记忆性T/B细胞是加重慢性排斥进程的重要原因，我们采用包括细胞治疗在内的联合方案，明显延长了预致敏移植物存活时间，为进一步增强MSCs治疗慢性排斥效果提供了实验依据。以上部分研究结果已发表研究论文。本课题为MSCs治疗肾移植慢性排斥提供了重要的实验依据和理论基础。 .