过表达GATA-4骨髓间充质干细胞通过外泌体修复心肌损伤的探讨

Exploration of GATA-4-overexpressing mouse bone marrow mesenchymal stem cells that repair myocardial infarction by secreting exosome

目的:探讨过表达GATA-4小鼠骨髓间充质干细胞(BMSC)通过分泌外排体(exosome)修复心肌损伤的机制。方法:通过慢病毒载体GV308携带GATA-4转染小鼠BMSC构建过表达GATA-4小鼠BMSC并加入基因开启剂——强力霉素(DOX),然后采用SBI公司的ExoQuick-TC提取分泌的exosome。通过电镜检测exosome的形态。经尾静脉注射采用Dir预染的过表达GATA-4-BMSCs分泌的exosome(过表达GATA-4组)、空载体-BMSCs分泌的exosome(空载体组)、BMSCs细胞分泌的exosome(BMSCs组),将不给予任何处理的心肌梗死(心梗)模型小鼠及正常喂养的小鼠分别设为未处理组和对照组。采用心脏彩超检测给予干预措施96h的心功能改善情况。进而采用小动物活体成像检测心梗局部Dir荧光强度。番茄凝集素评估心梗局部血管生成及采用免疫组化检测心梗局部C-kit阳性细胞的数量。结果:过表达GATA-4组较其他组可以更好地改善心梗后的心功能,射血分数及环比收缩改善幅度最大。过表达GATA-4组较其他组在96h时心脏局部可见更高荧光强度。番茄凝集素实验可见:过表达GATA-4组较其他组在给予干预措施后96h,可以表达出更多的血管。C-kit免疫组织化学检测可见:过表达GATA-4组较其他组在给予干预措施后96h,心梗局部C-kit细胞数量较其他组多。结论:过表达GATA-4的BMSCs可以通过分泌的exosome增强"归巢"效应、促进心梗局部血管增生、有效动员C-kit阳性细胞修复心肌损伤。

Objective：To test the hypothesis that GATA-4-overexpressing mouse bone marrow mesenchymal stem cells（BMSCs）to repair myocardial infarction by secreting.Method：Overexpression of GATA-4 was constructed with lentiviral vector GV308 carrying GATA-4 in BMSCs.Doxycycline（DOX）was added to induce the gene.Then,SBI ExoQuick-TC was applied to extract the exosome.Electron microscopy was performed to examine the morphology of the exosome.The exosomes secreted by the GATA-4-overexpressing BMSC,GATA-4-freevector-BMSCs,and BMSCs were injected into mice with myocardial infarction via the tail veins,while the control group did not get any treatment,all mice werefed with normal diet.Improvement in cardiac function at 96 h after intervention was evaluated with cardiac color Doppler detection.Moreover,the Dir fluorescence intensity at the site of myocardial infarction was detected by small animal living imaging.Angiogenesis at this site was evaluated using tomato lectin,and the number of C-kit positive cells at the site was detected with immunohistochemical test.Result：Compared with the exosomes secreted by the other BMSCs,the exosome secreted by GATA-4-overexpressing BMSCs significantly improved the cardiac function after myocardial infarction,with higher ejection fraction and better link-relative contraction.Higher fluorescence intensity was observed at the site of myocardial infarction at 96 h after application of the exosome secreted by GATA-4-overexpressing BMSCs.Results from the tomato lectin experiment indicated that more vessels grew in the group with GATA-4-overexpressing BMSCs than in the other groups at 96 h after intervention.Results from the C-kit immunohistochemical test indicated that the number of Ckit cells at the site of myocardial infarction was higher for the exosome secreted by GATA-4-overexpressing BMSCs than for the other exosomes at 96 h after intervention.Conclusion：GATA-4-overexpressing BMSCs can strengthen the ＂ homing＂ effect,promote angiogenesis at the site of myocardial infarction,and effectively mobilize C-kit positive cells to repair myocardial infarction.