过表达GATA-4骨髓间充质干细胞通过外排体修复心肌损伤的机制

发布时间：2019-03-25 07:57

【摘要】：目的:探讨过表达GATA-4小鼠骨髓间充质干细胞(BMSCs)通过分泌外排体(exosome)修复心肌损伤的机制。方法:构建过表达GATA-4的小鼠BMSCs并提取其分泌的exosome。而后将GATA-4-BMSCs-exosome与BMSCs共同培养,空载体-BMSCs-exosome与BMSCs共同培养,BMSCs-exosome与BMSCs共同培养、BMSCs单独培养及小鼠心肌细胞单独培养48h,采用Q-PCR定量检测心肌肌钙蛋白T(cTnT)、α肌动蛋白(α-actin)、连接蛋白43(connexin 43)、结蛋白(Desmin)心肌特异性抗原表达量。再将过表达GATA-4BMSCs分泌的exosome与心肌细胞共培养,空载体-BMSCs分泌的exosome与心肌细胞共培养,BMSCs分泌的exosome与心肌细胞共培养及心肌细胞单独用于低氧培养无血清培养诱导细胞凋亡作为阳性对照培养48h诱导细胞凋亡。采用流式细胞技术检测各组细胞的凋亡率。进一步采用TMT标记定量蛋白质组学分析过表达GATA-4小鼠BMSCs分泌exosome内有关细胞分化及抗凋亡的蛋白。结果:Q-PCR显示:过表达GATA-4-BMSCs分泌的exosome可以有效促进BMSCs向心肌细胞转化并具有极强的抗凋亡能力。蛋白质谱提示有8个蛋白有关细胞分化。有6个蛋白有关细胞调亡。其中Slit homolog 2protein及Connective tissue growth factor蛋白既涉及细胞凋亡又涉及细胞分化。结论:过表达GATA-4-BMSCs分泌的exosome可以促进BMSCs向心肌细胞分化并具有抗凋亡能力。Slit homolog 2protein及Connective tissue growth factor蛋白既涉及细胞凋亡又涉及细胞分化。
[Abstract]:Aim: to investigate the mechanism of over-expression of GATA-4 mouse bone marrow mesenchymal stem cells (BMSCs) in repairing myocardial injury by secreting efflux (exosome). Methods: the mouse BMSCs expressing GATA-4 was constructed and the exosome. secreted by it was extracted. Then GATA-4-BMSCs-exosome was co-cultured with BMSCs, empty vector-BMSCs-exosome was co-cultured with BMSCs, BMSCs was cultured alone and mouse cardiomyocytes were cultured for 48 h. The expression levels of cardiac troponin T (cTnT), 伪 actin 43 (connexin 43) and desmin (Desmin) myocardial specific antigen were detected by Q-PCR. The exosome secreted by GATA-4BMSCs was co-cultured with cardiomyocytes, and the exosome secreted by empty vector-BMSCs was co-cultured with cardiomyocytes. Exosome secreted by BMSCs was co-cultured with cardiomyocytes and cardiomyocytes were used alone in hypoxia culture to induce apoptosis in serum-free culture as a positive control culture for 48 hours. The apoptosis rate of each group was detected by flow cytometry. Furthermore, TMT labeled quantitative proteomics was used to analyze the proteins related to cell differentiation and anti-apoptosis in exosome secreted by over-expressed BMSCs in GATA-4 mice. Results: Q-PCR showed that over-expression of exosome secreted by GATA-4-BMSCs could effectively promote the transformation of BMSCs into cardiomyocytes and had strong anti-apoptosis ability. The protein profiles suggest that there are 8 proteins related to cell differentiation. There are six proteins involved in cell apoptosis. Among them, Slit homolog 2protein and Connective tissue growth factor proteins are involved in both apoptosis and differentiation. Conclusion: overexpression of exosome secreted by GATA-4-BMSCs can promote the differentiation of BMSCs into cardiomyocytes and has the ability of anti-apoptosis. Slit homolog 2protein and Connective tissue growth factor proteins are involved in both apoptosis and differentiation of cardiomyocytes.
【作者单位】：云南省第一人民医院心脏大血管外科;
【基金】：国家自然科学基金(No:81460073,31460298) 云南省科技厅-昆明医科大学应用基础研究联合专项(No:2014FB089) 云南省教育厅科学研究基金(No:2015Z051) 中国博士后科学基金(No:2015M582764XB) 成都医学院2015年度科研项目(No:CYZ15-18) 云南省医学后备人才(No:H-201607)
【分类号】：R542.2

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