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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。
AIM: To investigate the mechanism of GATA-4 mouse bone marrow mesenchymal stem cells (BMSC) repairing myocardium injury by secreting exosomes. METHODS: GATA-4 mouse BMSC was overexpressed by GATA-4 transfection in lentiviral vector GV308 and added to the gene opener-doxycycline (DOX), then excreted by SBI’s ExoQuick-TC exosome. Exosome morphology was examined by electron microscopy. Exosomes (empty vector group) secreted by empty vector-BMSCs and exosomes (BMSCs group) secreted by BMSCs were injected into the tail vein with Dir pre-stained overexpression GATA-4-BMSCs, , Myocardial infarction (MI) model mice without any treatment, and mice fed the normal diet were set as the untreated group and the control group, respectively. The improvement of cardiac function was detected by echocardiography 96 hours after intervention. Then, the local Dir fluorescence intensity of myocardial infarction was detected by live animal imaging. Tomato lectin was used to assess the local angiogenesis in myocardial infarction and the number of C-kit positive cells in myocardial infarction was detected by immunohistochemistry. Results: Compared with other groups, GATA-4 overexpressing group could improve the cardiac function after myocardial infarction. The ejection fraction and systolic contraction improved most significantly. GATA-4 overexpression in the 96h group compared with other groups showed higher local fluorescence intensity. Tomato lectin experiment shows that GATA-4 overexpression can express more blood vessels 96 h after intervention than other groups. C-kit immunohistochemistry showed that the number of C-kit cells in myocardial infarction was more than that in other groups at 96h after GATA-4 overexpression compared with other groups. Conclusion: GATA-4-overexpressing BMSCs can enhance the “homing” effect through the secretion of exosomes and promote the proliferation of myocardial infarction. It can effectively mobilize C-kit positive cells to repair myocardial injury.