间充质干细胞分泌的外泌体保护心梗后心肌通过p53介导的自噬通路

发布时间：2018-06-16 17:43

本文选题：间充质干细胞(MSCs) + 心肌梗死　；参考：《浙江大学》2017年硕士论文

【摘要】：背景:在全球范围内,心肌梗死是危害人类健康的重大疾病,心肌梗死导致的充血性心力衰竭是导致心血管事件死亡的主要原因。关于如何促进心肌梗死后心脏的组织修复和功能重建是心血管研究领域的研究热点和重大科学问题。有研究表明骨髓间充质干细胞(MSCs)移植能通过旁分泌的作用减少梗死周边区心肌细胞的凋亡、增加存活心肌,是心肌组织修复和功能重建的有效手段,而且间充质干细胞细胞来源丰富,移植后免疫反应几乎没有,但其治疗心肌梗死的分子生物机制尚未完全揭示。自噬是细胞对自身细胞器和代谢废物清除的一种进化上保守的重要功能。自噬对各种心血管疾病都有重要的作用。而且细胞自噬对细胞的存活具有重要的调控作用。近年来,围绕自噬在心血管稳态和疾病中的作用的研究方面取得了快速的进展。有研究表明在心脏缺血再灌注模型中,在缺血阶段自噬会增加,此时增强的自噬功能对心脏具有保护作用,而在再灌注阶段过度增高的自噬不利于心肌细胞的存活。当一些因素刺激细胞产生过度的自噬时,细胞会发生"自噬性细胞死亡",特点是胞浆中存在丰富的自噬泡。为了深入探讨间充质干细胞移植后能保护心肌细胞及改善心脏功能的作用和细胞自噬是否有关,我们在小鼠心肌梗死后移植间充质干细胞,进一步阐明自噬通路在干细胞治疗心梗上的作用机制。方法与结果:在体内实验部分,我们建立小鼠心肌梗死模型,并在心梗后在心肌内注射骨髓来源的间充质干细胞(MSCs)。之后监测小鼠1,3,7,14,28天的心功能和心脏重构的变化以及比较小鼠心脏组织自噬的改变。我们发现心梗后移植间充质干细胞确实能减少心梗后心肌细胞的凋亡和改善心脏功能。心梗后心脏组织的自噬水平上调,而在移植间充质干细胞后心脏组织的自噬水平下调。在体外实验部分,我们将间充质干细胞与乳鼠心肌细胞共培养,在缺氧培养箱中模拟心梗的条件,检测乳鼠心肌的自噬水平。我们发现缺氧24小时后心肌细胞的死亡率升高,自噬水平上调,而在干细胞共培养组中,心肌细胞死亡率减少,自噬水平下调。使用自噬抑制剂后,心肌细胞死亡率有所减少。缺氧组心肌细胞p53水平升高,心肌细胞线粒体膜电位降低,而在共培养组中,p53水平下降,心肌细胞线粒体膜电位较缺氧组有所升高。为了进一步研究间充质干细胞是通过分泌何种物质,进而调节心肌细胞的自噬水平。我们提取间充质干细胞的外泌体(exosomes),发现经外泌体共培养后的心肌细胞有相同的现象。其中mir-125b发挥了重要的作用。结论:在心梗模型中,间充质干细胞分泌外泌体,外泌体中的mir-125b作用于心肌细胞,通过p53/bnip3信号通路下调心肌细胞的自噬水平,减少心肌细胞的自噬性细胞死亡,从而改善心脏功能和减少心脏的重构。
[Abstract]:Background: myocardial infarction is a major disease that endangers human health worldwide. Congestive heart failure caused by myocardial infarction is the main cause of cardiovascular death. How to promote cardiac tissue repair and functional reconstruction after myocardial infarction is a hot and important scientific issue in cardiovascular research. Some studies have shown that bone marrow mesenchymal stem cells (MSCs) transplantation can reduce cardiomyocyte apoptosis and increase myocardial viability through paracrine, which is an effective means of myocardial tissue repair and functional reconstruction. Moreover, mesenchymal stem cells (MSCs) are abundant in origin and have little immune response after transplantation, but the molecular biological mechanism for the treatment of myocardial infarction has not been fully revealed. Autophagy is an important and evolutionarily conserved function of cells to clear their own organelles and metabolic wastes. Autophagy plays an important role in various cardiovascular diseases. Moreover, autophagy plays an important role in regulating cell survival. In recent years, rapid progress has been made on the role of autophagy in cardiovascular homeostasis and disease. Some studies have shown that autophagy increases in the ischemic phase, and the enhanced autophagy has protective effect on the heart, while excessive autophagy during the reperfusion phase is not conducive to the survival of cardiac myocytes. When some factors stimulate excessive autophagy, autophagic cell death occurs, characterized by abundant autophagy in the cytoplasm. In order to investigate whether the role of mesenchymal stem cells (MSCs) in protecting cardiomyocytes and improving cardiac function is related to autophagy, we transplanted mesenchymal stem cells (MSCs) after myocardial infarction in mice. To further elucidate the mechanism of autophagy pathway in stem cell therapy of myocardial infarction. Methods and results: in vivo, we established myocardial infarction model in mice and injected MSCs derived from bone marrow into myocardium after myocardial infarction. The changes of cardiac function and cardiac remodeling and the changes of autophagy in heart tissue were monitored after 1428 days. We found that transplantation of mesenchymal stem cells after myocardial infarction can reduce myocardial cell apoptosis and improve cardiac function. Autophagy was up-regulated in cardiac tissue after myocardial infarction and down-regulated after transplantation of mesenchymal stem cells. In vitro, mesenchymal stem cells (MSCs) were co-cultured with neonatal cardiomyocytes to simulate myocardial infarction in anoxic incubator. We found that cardiomyocyte mortality increased and autophagy increased 24 hours after hypoxia, whereas in stem cell co-culture group, myocardial cell mortality decreased and autophagy decreased. Cardiac myocyte mortality was reduced after the use of autophagy inhibitors. In hypoxia group, the level of p53 increased and the mitochondrial membrane potential of cardiomyocytes decreased, while in co-culture group, the level of p53 decreased, and the mitochondrial membrane potential of cardiomyocytes was higher than that of hypoxia group. In order to further study how mesenchymal stem cells secrete substances to regulate cardiac myocyte autophagy level. Exocrine exosomeses of mesenchymal stem cells were extracted and the same phenomenon was found in cardiomyocytes after co-culture of exocrine mesenchymal stem cells. Mir-125b plays an important role. Conclusion: in myocardial infarction model, mesenchymal stem cells secrete exocrine and mir-125b acting on cardiac myocytes, down-regulate autophagy level and decrease autophagic cell death through p53/bnip3 signaling pathway. Thus improving heart function and reducing heart remodeling.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22

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