肌成纤维细胞来源外泌体介导主动脉缩窄后心脏重构的作用

Effects of myofibroblasts-derived exosomes on cardiac remodeling post transverse aortic constriction

目的:观察肌成纤维细胞来源外泌体对主动脉缩窄后心脏重构的影响。方法:利用促纤维化因子转化生长因子-β(TGF-β)诱导心脏成纤维细胞活化,将原代心肌细胞分为正常组、PBS组、外泌体组,通过超高速离心分离提取成纤维细胞上清液中的外泌体,并将其与外泌体组心肌细胞共同孵育48 h。实时定量聚合酶链反应及免疫荧光染色法检测心肌细胞肥厚指标(心房利钠肽、脑钠肽和β肌球蛋白重链)以及心肌细胞表面积。选取C57雄性小鼠32只,随机分为对照组、假手术组、主动脉缩窄-对照组、主动脉缩窄-外泌体组,每组8只。主动脉缩窄组小鼠进行主动脉缩窄模型构建,外泌体组小鼠每日通过尾静脉注射20µg外泌体,连续注射4周。术后8周行心脏超声检测各组小鼠心功能状态,麦胚凝集素(WGA)染色检测小鼠左心室心肌肥厚程度。结果:电镜下提取物呈典型杯口状结构,Westrtn blot显示该物质含有外泌体特有标志物CD63、TSG101。相对于正常组及PBS组,外泌体组心肌细胞心房利钠肽、脑钠肽和β肌球蛋白重链的mRNA表达水平均显著升高,心肌细胞表面积显著增加(P均<0.05)。与对照组、假手术组、主动脉缩窄-对照组相比,主动脉缩窄-外泌体组小鼠射血分数、短轴缩短率均显著下降(P均<0.05),左室收缩末期内径、左室舒张末期内径均显著增加,心肌细胞表面积显著增加(P均<0.05)。结论:激活状态下成纤维细胞来源外泌体可以促进心肌细胞病理性肥厚的发生,加速主动脉缩窄后心脏重构的进程。

Objective:To explore the role of exosomes derived from myofibroblasts in cardiac remodeling post transverse aortic constriction(TAC).Methods:Exosomes isolated from supernatant of cardiac fibroblasts(CFs)stimulated by TGF-βwere incubated with cardiomyocytes for 48 h.PCR and immunofluorescence assay were used to assess the expression levels of hypertrophic-related gene,atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain(β-MHC)and cardiomyocyte cell size.Additionally,thirty-two male mice were divided into 4 groups as control group,sham group,TAC-NC group(mice underwent TAC procedure and injected with PBS through tail vein),and MI+Exos-activated group(mice underwent TAC procedure and injected with esosomes derived from myofibroblasts through tail vein),with 8 mice in each group.After 8 weeks,cardiac function and the extent of cardiac hypertrophy were assessed by echocardiography and WGA-FITC staining.Results:Transmission electron microscopic image showed that myofibroblasts-derived exosomes have the characteristic round or cup-shaped delineated by a lipid bilayer.The results of western blot analysis demonstrated the expression of CD63 and tumor susceptibility gene 101(TSG101)in all exosome markers.Compared with control and PBS groups,the expression levels of ANP,BNP,β-MHC mRNA were increased significantly,and relative surface was also enlarged in exosome group(all P<0.05).Compared with control group,sham group and MI+Exo-activated group,the ejection fraction and short-axis shortening were significantly decreased in TAC-NC group,while the LVESD and LVEDD were significantly increased(all P<0.05).And the surface of myocardial cell was significantly increased(P<0.05).Conclusion:Activated fibroblast-derived exosomes can promote the occurrence of pathological hypertrophy of cardiomyocytes and accelerate the process of cardiac remodeling after TAC.