Islet-1修饰MSCs来源外泌体通过mTOR通路修复受损心肌细胞的机制研究

MSCs differentiation into myocardial cells to replace the damaged myocardial cells has been a major breakthrough, but it is difficult to satisfied the urgent needs of acute myocardial infarction. Researches indicated that MSCs was able to restore some of the cardiac function through paracrine in 72h, the exosomes were considered to be the main material to repair function, but the mechanism were not clear. Our previous study showed that MSCs over-expression of Islet-1 could significantly reduce the apoptosis of hypoxia-ischemia myocardial cells after indirect co-culture, and the proliferation associated mTOR pathway of myocardial cells was activated. Therefore, we put forward the hypothesis: MSCs over-express Islet-1 could improve the mTOR or related-miRNA carrying capacity of exosomes, the mTOR pathway of myocardial cells was activated after myocardial cells intake these exosomes and maintain their survival in hypoxia-ischemia environment. This study was proposed survey the status of mTOR and related-miRNA carrying capacity of exosomes which secreted from Islet-1 over-expressed MSCs and discuss whether the myocardial cells maintaining their survival in hypoxia-ischemia environment by intaking these exosomes. This study in order to reveal the mechanism of exosomes myocardial repair function which secreted from MSCs, to provide important theoretical basis for application of MSCs in early treatment of myocardial infarction.

MSCs分化为心肌细胞以替代受损心肌在临床上虽已有较大突破，但因诱导方法和分化时间的局限性，使其难以满足临床的迫切需求。研究指出MSCs能通过旁分泌在72h内恢复部分心肌功能，其中外泌体因能携带多种蛋白、mRNA及miRNA被认为是发挥修复功能的主要物质，但具体机制仍不明了。我们前期研究表明Islet-1修饰的MSCs与缺血缺氧心肌细胞间接共培养后能显著降低其凋亡，且心肌细胞中与增殖相关的mTOR通路被激活，因此我们提出假设：Islet-1能提高MSCs来源外泌体mTOR或相关miRNA携带量，被受体心肌摄入后激活mTOR通路维持自身存活。本研究拟通过观察Islet-1修饰MSCs来源外泌体携带mTOR及相关miRNA情况，探讨受体心肌细胞是否通过摄入外泌体而激活自身mTOR通路以维持存活，从而揭示MSCs通过外泌体修复受损心肌的相关机制，为MSCs应用于心肌损伤早期治疗提供重要理论依据。

心脏是严重脓毒症/脓毒症休克的重要靶器官，其功能障碍在脓毒症中表现为多种不同的方式，包括收缩期或舒张期左心室和/或右心室功能障碍，心排血量和供氧不足，原发性心肌细胞损伤等。脓毒症诱发心功能障碍可使病死率增加50%，应引起我们足够的重视。本项目发现，脓毒症过程中心肌细胞线粒体线粒体NCLX介导的Ca2+流出显著降低，这可能是引发心肌细胞线粒体钙超载，造成心肌损伤的重要因素。同时，我们发现huMSCs分泌的外泌体可以恢复脓毒症造成的心肌细胞线粒体Ca2+流出障碍，并因此降低线粒体损伤。进一步研究其机制发现，huMSCs外泌体中携带这比大量Pink1 mRNA， 用huMSCs外泌体处理CLP小鼠后，Pink1 mRNA可从huMSCs外泌体中转移到受体心肌细胞，增加其Pink1表达。PINK1是NCLX介导的Ca2+外排的重要调控因子，因此huMSCs外泌体的处理能够通过恢复NCLX活性，增加脓毒症过程中心肌细胞线粒体Ca2+流出，防止线粒体发生钙超载，保护线粒体，最终发挥心肌保护作用。本项目为实现间充质干细胞来源的外泌体防止脓毒症引发心肌受损的临床应用打下了重要的实验基础，对临床上解决脓毒症引发的心脏功能障碍、降低脓毒症患者死亡率、改善患者预后具有重要的研究意义。