过表达GATA-4小鼠骨髓间充质干细胞通过分泌外泌体增强其抗凋亡能力的研究  

GATA-4 Overexpressed Mouse Bone Marrow Mesenchymal Stem Cells Increase Anti-apoptosis by Secreting Exosome

作　　者：贺继刚[1] 李贝贝 韩金秀 李洪荣[1] 撒亚莲[1] 严丹[2] 杨晓华[2] HE Ji-gang;LI Bei-bei;HAN Jin-xiu;LI Hong-rong;SA Ya-lian;YAN Dan;YANG Xiao-hua(Department of Cardiac and Macrovascular Surgery,the First People's Hospital of Yunnan Province,Kunming 650032,China;Intensive Care Unit,the First People's Hospital of Yunnan Province,Kunming 650032,China)

机构地区：[1]云南省第一人民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重症医学科,云南省昆明市650032

出　　处：《中国全科医学》2018年第24期2961-2966,共6页Chinese General Practice

基　　金：国家自然科学基金资助项目(81460073;31460298);云南省科技厅--昆明医科大学应用基础研究联合专项资金项目(2014FB089);云南省教育厅科学研究基金(2015Z051);中国博士后科学基金(2015M582764XB);成都医学院2015年度科研项目(CYZ15-18);云南省医学后备人才(H-201607)

摘　　要：目的探讨过表达GATA-4小鼠骨髓间充质干细胞(BMSCs)是否通过分泌外泌体(Exosome)增强其细胞抗凋亡能力,进而提高心肌梗死后心功能。方法 2017年1—12月选取健康4周龄SPF级C57BL/6小鼠45只,通过慢病毒载体GV308携带GATA-4转染小鼠BMSCs构建过表达GATA-4小鼠BMSCs并加入基因开启剂强力霉素(DOX),而后采用Exo Quick-TC提取BMSCs分泌的Exosome,并检测Exosome纯度、电镜下观察其形态。体外实验:与过表达GATA-4 BMSCs分泌的Exosome共同培养的心肌细胞为A组(体外),与空载体BMSCs分泌的Exosome共同培养的心肌细胞为B组(体外),与BMSCs分泌的Exosome共同培养的心肌细胞为C组(体外),低氧(1%)无血清条件下培养的心肌细胞为D组(体外),正常条件下单独培养的心肌细胞为E组(体外),各组均培养48 h,后采用流式细胞术检测各组心肌细胞凋亡率,采用Western blotting法检测Caspase-3、Caspase-8、Caspase-9、细胞色素C表达水平。体内实验:选取12只小鼠建立心机梗死模型,造模成功48 h后经尾静脉注射80 000μg的Exosome,其中A组(体内)注射过表达GATA-4 BMSCs分泌的Exosome,B组(体内)注射空载体BMSCs分泌的Exosome,C组(体内)注射BMSCs分泌的Exosome,另取3只心肌梗死未处理小鼠作为D组(体内),3只正常小鼠作为E组(体内)。于注射Exosome后48 h采用心脏超声仪(PHILIPS EPIQ 7C)评估各组小鼠心功能。完成心脏彩超后采用原位免疫组化法评估心肌梗死小鼠心脏凋亡细胞数量。结果 Exosome表达量为0.272 5,Exosome纯度为5.25×108个,电镜可见Exosome大小40~100 nm。A组(体外)细胞凋亡率低于B组(体外)、C组(体外)、D组(体外),但高于E组(体外)(P<0.05)。A组(体外)Caspase 8、细胞色素C表达水平低于B组(体外)、C组(体外)、D组(体外)及E组(体外)(P<0.05)。A组(体内)小鼠射血分数(EF)、环比收缩(FS)前后差值高于B组(体内)、C组(体内)、D组(体内)及E组(体内)(P<0.05)。A组(体内)小鼠心肌细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 infarc

关 键 词：心肌梗死 干细胞 外泌体 

分 类 号：R542.22[医药卫生—心血管疾病]