关键词: 心肌梗死, 干细胞, 外泌体

Abstract: Objective　To explore the role of GATA-4 overexpressed mouse bone marrow mesenchymal stem cells （BMSCs） in the improvement of cardiac function after myocardial infarction repair by enhancing the anti-apoptotic capability of BMSCs via secreting Exosome.Methods　This study was conducted between January and December 2017.60 healthy SPF C57BL/6 mice aged 4 weeks were selected.GATA-4 overexpressed mouse BMSCs were constructed by transfecting the BMSCs with lentiviral vector GV308 carrying GATA-4.Doxycycline was added to induce the gene.Then，SBI ExoQuick-TC was adopted to extract the Exosome secreted by the transfected BMSCs.Exosome purity was determined by SBI Exosome Antibodies，Array，and ELISA Kits and observed by an electron microscope.In vitro experiments：coculture was performed between Exosome secreted by GATA-4 overexpressed BMSCs and myocardial cells（group A），between Exosome secreted by GATA-4-free-vector-BMSCs and myocardial cells（group B），and between Exosome secreted by BMSCs and myocardial cells （group C）．For the positive control group（group D），myocardial cells alone were subjected to hypoxia culture and serum-free culture to induce apoptosis.Meanwhile，for the negative control group（group E），myocardial cells alone were cultured under normal conditions.All these 5 groups were cultured for 48 h to induce apoptosis.The cell apoptosis rate was determined through flow cytometry，and Caspase-3，Caspase-8，Caspase-9，and cytochrome C were measured through Western blot.In vivo experiments：48 h after the establishment of mouse model of myocardial infarction in 30 mice，Exosomes secreted by GATA-4 overexpressed BMSC，and GATA-4-free-vector-BMSCs and BMSCs were injected into the tail veins of group A，group B，group C，respectively.Treatment-free 3 mice models of myocardial infarction（group D） and 3 normal mice receiving normal feeding（group E） were used as the control groups.The cardiac function was evaluated by Philips EPIQ 7 C Cardiac Ultrasound at 48 h after intervention.After that，the number of apoptotic cells in myocardial infarction focus was detected through immunohistochemistry with in situ hybridization methods.Results　In vitro experiments：Electron microscopy showed that the size of Exosome ranged from 40 nm to 100 nm，and expression amount of Exosome was 0.272 5，the purity of Exosome was 5.25×108 .Flow cytometry results showed that the proportion of apoptotic cells in myocardial infarction focus after intervention and adoption for 48 h in group A was lower than that of groups B，C，D，but higher than that of group E（P<0.05）．Western blotting results indicated that the expression levels of Caspase 8 and cytochrome C in group A were lower than those of other groups（P<0.05）．In vivo experiments：Cardiac ultrasound results revealed that group A had better EF and FS D-value than other groups（P<0.05）．Analysis of immunohistochemical examination with in situ hybridization showed that the number of apoptotic cells in myocardial infarction focus after intervention and adoption for 48 h in group A was more than that of group E，but less than that of groups B，C and D（P<0.05）．Conclusion　Exosome secreted by GATA-4 overexpressed mouse BMSCs can enhance the anti-apoptotic capability of BMSCs to effectively improve the cardiac function after myocardial infarction.

Key words: Myocardial infarction, Stem cells, Exosomes