间充质干细胞移植对急性心肌梗死小鼠单核细胞M1/M2亚型转化作用及机制实验研究

The results of the applicant and the others’ previous researches showed that less survival and too short time of transplanted MSCs stayed in the infarct tissue，which leads to be doubted that grafted MSCs in the myocardium dominate the M1/ M2 subtype transformation to inhibit inflammatory injury, promote cardiac repair in the treatment of acute myocardial infarction..In this study,the survival of grafted cells is traced dynamically in spleen, bone marrow etc beyond heart by real time fluorescence quantitative polymerase chain reaction for detection of human GAPDH gene and fluorescence microscopy,after transplantation of MSCs obtained from umbilical cord blood in mice with acute myocardial infarction.To explore the effects of MSCs on M1/M2 subtype conversion of extra-cardiac monocyte including blood,spleen and bone marrow/ cardiac macrophage is acted as a focal point.The conversion of M1/M2 subtypes in extra-cardiac monocyte and cardiac macrophage，inflammation and cardiac remodeling, function as observation indexes.The cell factors derivated from stem cell or the related inhibitors as the means of intervention. The multi-levels research are carried out by in vitro effects observed, and also in vivo effects of validation, and then to explore the mechanisms in vitro. This study is aimed to elucidate the effect of MSCs transplantation on monocyte M1/M2 subtype transformation, and to explore the mechanisms possible for monocyte M1/M2 subtype transformation mediated by MSCs, and also clarify curative effect of MSCs transplantation in acute myocardial infarction does not depends only on the MSCs survival in myocardium,and that MSCs grafted in the organs beyond the heart also has an important role; This study is to explore the new mechanism for MSCs transplantation,and also a new important extension for the mechanism for MSCs transplantation in the treatment of myocardial infarction.

基于申请人前期工作与相关研究发现对急性心梗间充质干细胞（MSCs）移植，MSCs在心肌组织定植时间过短及数量过少。我们对心肌组织内MSCs主导巨噬细胞M1/ M2亚型转化是MSCs抑制心梗后炎性损伤、促心脏修复的主要机制产生质疑。本研究采用实时荧光定量PCR检测人GAPDH基因及病理技术，动态追踪人脐血提取的MSCs移植治疗急性心梗小鼠后MSCs在脾、骨髓等心外存活状况。以MSCs对外周血、脾、骨髓等心外单核细胞M1/ M2亚型转化为切入点，以炎性心肌损伤及心脏重构功能等为观察指标，以细胞因子或相应抑制剂等为干预手段，通过体外效应观察、体外机制探讨、再到体内效应验证。旨在阐明MSCs促单核细胞M1/M2亚型转化作用及其机制，明确急性心梗MSCs移植疗效不仅仅依赖定植心梗组织MSCs，迁移致心外MSCs亦有重要的治疗效应；本研究是对MSCs移植治疗心梗机制新的探索及重要拓展。

本课题主要观察小鼠心肌梗死心内-心外以单核巨噬细胞M1/M2亚型转化为主的炎症反应及脐血间充质细胞（hUCB-MSCs）调节验证双重反应一定程度改善心肌修复的过程。对急性心梗造模后的小鼠进行尾静脉注射hUCB-MSCs及GFP标记的hUCB-MSCs，在心梗后第3、7、14天在小鼠各实质器官中追踪GFP-hUCBMSCs, Masson染色计算梗死区域中剩余心肌和胶原体积分数（CVF）的比例，流式细胞术和western blot以分析心内外系统M1 / M2亚型及其比例， 免疫组化及ELISA测定心梗组织和血清IL-10、IDO、PGE2水平，14天时超声心动图检测心功能。结果：hUCBMSCs未定植心脏中，且hUCBMSCs可促使单核巨噬细胞向抗炎修M2亚型转化，降低心梗后小鼠心内-外全身系统的炎症损伤因子水平，降低胶原体积分数，提高LVEF，一定程度上保护残余心肌并改善心功能。结论：hUCB-MSCs不是在心梗部位分化为心肌细胞，而是通过调节心内-心外单核巨噬细胞M1/M2亚型转化改善炎症双重效应，减轻炎症损伤，促进心脏修复。