过表达MIF的人源MSCs分泌的外泌体抑制心肌细胞衰老的机制研究

The protection of exosomes (Exo) secreted from human mesenchymal stem cells (MSCs) with over-expression macrophage migration inhibitory factor (MIF) for ischemic cardiomyocites is paid more and more attention. The study of the role of Exo secreting from MSCs with gene modification in myocardial protection is very important. Pre-experimental of this study discovered that Exo secreted from hMSCs with over-expressing MIF inhibited senescence of cardiomyocytes by AMPK pathway; Altogether 111 Exo's differential expression miRNAs were screened by high-throughput sequencing. The biological information method combined with the literature analysis shows that has-miR-126-3p plays an important role. We hypothesized that the Exo-has-miR-126-3p secreted by the hMSCs with over-expressing MIF could protect ischemic myocardium by regulating AMPK pathway. According to the hypothesis of this study, we are going to further research the regulation mechanism of Exo-has-miR-126-3p to inhibit the senescence of cardiomyocytes; and research the effects of Exo-has-miR-126-3p on myocardial protection and its mechanism in ischemic rats. The results of this study will provide new strategy for the treatment of ischemic heart disease.

骨髓间充质干细胞（MSCs）分泌的外泌体（Exo）对缺血心肌保护作用越来越受到重视。对间充质干细胞进行基因修饰分泌的Exo作用如何值得研究。本课题预实验发现过表达巨噬细胞移动游走抑制因子（MIF）的人源MSCs（hMSCs）分泌的Exo能抑制心肌细胞衰老；并初步证实AMPK信号通路参与这一过程；高通量测序筛选出Exo的差异表达miRNAs111个，生物信息分析结合文献报道显示has-miR-126-3p起重要作用。我们提出研究假设是过表达MIF的hMSCs分泌的Exo-has-miR-126-3p通过调节AMPK通路抑制心肌细胞衰老，对缺血心肌起保护作用。围绕研究假设，本课题拟进一步研究Exo-has-miR-126-3p抑制心肌细胞衰老的调控机制；Exo-has-miR-126-3p对在体缺血心肌保护作用及其机制。本课题研究成果将对缺血性心肌病治疗提供新思路。

外泌体移植是一种很有前途的无细胞治疗方法，用于缺血性心肌病治疗。本研究的目的是探讨来源于巨噬细胞迁移抑制因子(Macrophage migration inhibitory factor, MIF)修饰的脐带MSCs (umbilical cord MSCs, uMSCs)的外泌体在急性心肌梗死（acute myocardial infarction, AMI）大鼠模型中是否表现出优越的心脏保护作用，并揭示其潜在机制。我们从uMSCs (MSC-Exo)、MIF修饰的uMSCs (MIF-Exo)和MIF下调uMSCs (siMIF-Exo)分离出外泌体，并进行鉴定。研究这些外泌体在缺血/氧血(H/SD)条件下对人脐静脉内皮细胞(HUVEC)和 H9C2心肌细胞的保护作用。与MSC-Exo和siMIF-Exo相比，MIF-Exo在体外显著增强HUVEC的增殖、迁移和血管生成，并抑制H/SD条件下H9C2心肌细胞的凋亡。MIF-Exo还显著抑制心肌细胞凋亡，减少纤维化面积，并改善心脏功能，超声心动图测量梗死大鼠在体内。外泌体miRNA测序和qRT-PCR证实MIF-Exo中miRNA-133a-3p显著增加。在MIF-Exo和miR-133a-3p 抑制剂的作用下，HUVEC和H9C2心肌细胞的生物学效应减弱。这些效应随着siMIF-Exo和miR-133a-3p模拟物的孵育而加剧，这些模拟物增加了这些细胞中AKT蛋白的磷酸化。因此，MIF-Exo在体内外可以通过促进血管生成、抑制细胞凋亡、减少纤维化、保护心脏功能等方面发挥心脏保护作用。MIF-Exo的生物学活性机制涉及miR-133a-3p和下游AKT信号通路。本研究将为缺血性心肌病治疗提供新策略。