心肌成纤维细胞来源的exosome通过microRNA参与糖尿病心肌病发病的作用及机制研究

Diabetic cardiomyopathy (DCM) is one of the major causes of death in T2DM patients. The main pathological changes of DCM are cardiomyocytes hypertrophy, apoptosis and interstitial fibrosis. Exosome is one kind of extracellular vesicles containing miRNA, lncRNA, circRNA, mRNA and proteins, which could be uptaked by other kinds of cells. Thus exosomes play an important role in cellular communication. It is reported that CF exosomes mediated cardiac hypertrophy via miR-21_3p. And CF exosomes were also enriched of miR-30c, miR-133a, miR-34c, miR-125a-5p, miR-214 and miR-320 which were closely related to cardiomyocytes hypertrophy, apoptosis and cardiac fibrosis. Our primary data showed that high glucose could enhance the production of exosomes from cardiac fibroblast (CF). Exosomes derived from high glucose or high insulin treated CF were of stronger pro-hypertrophic effect in cardiomyocytes. And exosomes derived from high glucose treated CF also induced significant cardiomyocytes apoptosis in vitro. Given this, we speculate that high glucose and high insulin perhaps could change the level of the above microRNAs in CF exosomes, resulting in DCM. To verify the hypothesis, effects of high glucose and insulin on exosomal size, yield, miRNA expression would be determined. Then we would study the effects of CF exosomes inhibition on DCM-induced cardiac remodeling and dysfunction in diabetic cardiomyopathy model mice, which might be new strategy for prevention and treatment of DCM.

糖尿病心肌病（DCM）的主要病理改变为心肌细胞肥大、凋亡和心肌纤维化。据报道心肌成纤维细胞(CF)分泌的exosome含有miR-21_3p、miR-30c、miR-133a、miR-34c、miR-125a-5p、miR-214及miR-320等多种与DCM相关的miRNA。我们的前期数据显示：高糖能增加CF exosome 分泌量；CF在高糖、高胰岛素条件下分泌的exosome促肥大能力更强；CF exosome促心肌细胞凋亡。鉴于此，我们推测“高糖、高胰岛素引起的CF exosome的分泌量、miRNA表达谱的改变很可能是糖尿病心肌病发病的重要机制”。为验证该假说，我们将观察上述培养条件对CF exosome的形态、分泌量、miRNA表达谱以及促心肌细胞肥大、凋亡作用的影响；观察抑制CF exosome分泌能否改善糖尿病心肌病小鼠心脏病理改变及心功能异常，为防治DCM提供新策略。

糖尿病心肌病是2型糖尿病患者的重要死因，其主要病理改变为心肌细胞肥大、凋亡和心肌纤维化。Exosome(外泌体)是一种多数细胞都能分泌的细胞外囊泡，可被别的细胞所摄取；外泌体含有miRNA等物质，可以发挥调控细胞功能的作用。我们推测“高糖引起的心肌成纤维细胞（CF）外泌体的分泌量、miRNA表达谱的改变引起了心肌细胞miRNA表达改变，并且可能是糖尿病心肌病发病的重要机制”。. 我们发现高糖可以增加心肌成纤维细胞外泌体分泌量。我们分别将正常（低糖，LG）、高糖（HG）等培养条件下乳大鼠心肌成纤维细胞产生的外泌体（10ug/ml）与乳大鼠心肌细胞共培养24小时，发现高糖CF外泌体诱导心肌肥大的能力强于正常外泌体。miR-21-3p是一种能够促进心肌细胞肥大生长的miRNA，SORBS2是miR-21-3p的靶蛋白，与心肌肥大相关。我们发现高糖CF外泌体中miR-21-3p的表达量是正常CF外泌体的4倍。然后我们将高糖CF外泌体（10ug/ml）、miR-21-3p inhibitor（100nM）与乳大鼠心肌细胞共培养48 小时。通过亮氨酸掺入实验发现抑制miR-21-3p能够消除高糖CF外泌体促进心肌细胞肥大的能力。我们通过高脂喂养加链脲佐菌素注射的方式建立了糖尿病模型小鼠，发现尾静脉注射miR-21-3p的敲减腺相关病毒能够降低糖尿病小鼠的心肌细胞肥大，但不明显降低心脏重量。. 通过分析上述数据，我们得出了以下结论：高糖除了可以直接促进心肌细胞肥大，还可以通过外泌体这一旁分泌机制放大其促心肌细胞肥大的能力。高糖刺激的心肌成纤维细胞分泌的外泌体含有更高丰度的miR-21-3p，所以促心肌细胞肥大的能力强于正常心肌成纤维细胞的外泌体，参与了糖尿病心肌病过程中的心肌肥大的发生、发展。该研究可以为治疗糖尿病心肌病提供新的方向和新思路。..