过表达GATA-4骨髓间充质干细胞分泌的外泌体促进骨髓间充质干细胞向心肌样细胞分化  

GATA-4-overexpressed bone marrow mesenchymal stem cell secreted exosomes promote bone marrow mesenchymal stem cell differentiation into cardiomyocyte-like cells

作　　者：贺继刚[1] 王梓豪 谢巧丽 李洪荣[1] 严丹[2] 李永武[1] He Jigang;Wang Zihao;Xie Qiaoli;Li Hongrong;Yan Dan;Li Yongwu(Department of Cardiovascular Surgery,the First People’s Hospital of Yunnan Province,Kunming University of Science and Technology Affiliated Hospital,Kunming 650032,Yunnan Province,China;Department of Intensive Care Unit,the First People’s Hospital of Yunnan Province,Kunming University of Science and Technology Affiliated Hospital,Kunming 650032,Yunnan Province,China)

机构地区：[1]云南省第一人民医院,昆明理工大学附属医院心脏大血管外科,云南省昆明市650032 [2]云南省第一人民医院,昆明理工大学附属医院重症医学科,云南省昆明市650032

出　　处：《中国组织工程研究》2020年第19期2965-2971,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81460073)，项目负责人：贺继刚;云南省科技厅-昆明医科大学应用基础研究联合专项(2014FB089)，项目负责人：贺继刚;云南省教育厅科学研究基金(2015Z051)，项目负责人：贺继刚;中国博士后科学基金(2015M582764XB)，项目负责人：贺继刚;成都医学院2015年度科研项目(CYZ15-18)，项目负责人：贺继刚;云南省医学后备人才(H-201607)，项目负责人：贺继刚~~

摘　　要：背景:前期证明,过表达心脏基因转录因子GATA-4小鼠骨髓间充质干细胞分泌的外泌体(BMSCs GATA-4-exosome)可以促进BMSCs向心肌样细胞分化,提示其可以修复心肌梗死,另外还发现BMSCs GATA-4-exosome中及心肌梗死局部心肌组织中有miRNA-673-5p明显高表达且功能涉及细胞分化,可能是BMSCs GATA-4-exosome修复心肌梗死的关键分子。目的:探讨BMSCs GATA-4-exosome促进BMSCs向心肌样细胞分化的分子调控网络。方法:在小鼠BMSCs培养体系内加入miRNA-673-5p模拟物(miR-673-5p-mimic)作为实验组(BMSCs miR-673-5p-mimic),将BMSCs GATA-4组、BMSCs GATA-4-空载体组、BMSCs组、BMSCs miR-673-5p-inhibitor组作为混杂因素对照组,提取各组分泌的外泌体与BMSCs直接共培养24 h,采用免疫荧光定性检测和RT-PCR定量检测各组BMSCs中心肌特异性分子α-actin、Desmin、cTnT、Cx43的表达。根据microRNA靶基因预测结果,采用Western blot检测miRNA-673-5p对应靶基因TSC-1、ERK1/2及Mef2c转录蛋白表达。结果与结论:BMSCs miR-673-5p-mimic-exosome+BMSCs组荧光强度最强,心肌细胞特异性分子α-actin、Desmin、cTnT、Cx43的表达最高(P<0.05),TSC-1的表达最低(P<0.05)。结果表明BMSCs GATA-4-exosome通过miRNA-673-5p抑制TSC-1蛋白表达促进BMSCs向心肌样细胞分化。BACKGROUND:Preliminary study has shown that overexpression of GATA-4-overexpressing bone marrow mesenchymal stem cell(BMSCs)exosomes(BMSCsGATA-4-exosome)can promote BMSCs differentiate into cardiomyocytes,indicating it can repair myocardial infarction.Additionally,high expression of miRNA-673-5p is observed in miRNA-673-5p BMSCsGATA-4-exosome and focal myocardium of myocardial infarction,which involve in cell differentiation,suggesting that miRNA-673-5p may be a key molecular of BMSCsGATA-4-exosome for repairing myocardial infarction.OBJECTIVE:To investigate the molecular regulatory network of BMSCsGATA-4-exosomes that promotes BMSCs differentiation into cardiomyocyte-like cells.METHODS:miR-673-5p-mimic was added to the BMSCs culture system as an experimental group(BMSCsmiR-330-3p-mimic).BMSCsGATA-4,BMSCsGATA-4-empty vector,BMSCs and BMSCsGATA-4-miR-673-5p-inhibitor groups were set as confounding factor control groups.The exosomes and myocardial cells secreted by each group were co-cultured for 24 hours.The expression levels of myocardium specific moleculesα-actin,Desmin,cTnT and Cx43 were detected by immunofluorescence and RT-PCR.The expression levels of the corresponding miRNA-673-5p target genes TSC-1,ERK1/2 and Mef2c were detected through western blot assay based on the prediction results of the microRNA target gene.RESULTS AND CONCLUSION:The BMSCsmiR-673-5p-mimic-exosome+BMSCs culture group had the highestα-actin,Desmin,cTnT and Cx43 levels(P<0.05),and the lowest TSC-1 expression(P<0.05).In summary,BMSCsGATA-4-exosome inhibits the expression of TSC-1 via miRNA-673-5p to promote BMSCs differentiation into cardiomyocyte-like cells.

关 键 词：miRNA-673-5p TSC-1蛋白 GATA-4 外泌体 小鼠 骨髓间充质干细胞 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]