摘要
目的探讨过表达GATA-4小鼠骨髓间充质干细胞(BMSCs)修复心肌损伤的机制。方法于2015年3月—2016年12月,选取60只健康4周龄SPF级雄性C57BL/6小鼠,分离BMSCs,采用流式细胞检测CD29、CD44、CD11b、干细胞抗原1(SCA-1)表达率。通过慢病毒载体GV308携带GATA-4转染小鼠BMSCs,构建过表达GATA-4-BMSCs,并加入基因开启剂多西环素(DOX),采用反转录聚合酶链反应(RT-PCR)方法检测转染72 h后过表达GATA-4-BMSCs中GATA-4 m RNA表达水平,并以未转染为阴性对照组。建立小鼠心肌梗死模型,经尾静脉注射BMSCs(n=3)、空载体-BMSCs(n=3)、过表达GATA-4-BMSCs(n=3)500μl,并将心肌梗死未处理组(n=3)及正常小鼠(n=3)同时给予喂食含有DOX的液体作为对照1组及对照2组。通过心脏彩超检测给予干预措施72 h后心功能改善情况。选取正常小鼠正常喂养(n=3)作为对照3组。采用小动物活体成像检测心脏区域荧光强度,番茄凝集素评估心肌梗死局部心肌血管数量,免疫组化检测心肌梗死局部C-kit阳性细胞数量。结果小鼠BMSCs CD29表达率为98.0%,CD44表达率为100.0%,CD11b表达率为0.1%,SCA-1表达率为99.5%。过表达GATA-4-BMSCs组GATA-4 m RNA表达水平为(78.17±1.32),高于阴性对照组的(0.57±0.34)(t=2.576,P<0.05)。各组小鼠射血分数(EF)、环比收缩(FS)变化值比较,差异均有统计学意义(P<0.05);其中过表达GATA-4-BMSCs组EF、FS改善幅度高于对照1组、对照2组、BMSCs组和空载体-BMSCs组(P<0.05)。各组小鼠心脏区域荧光强度、心肌血管数量、C-kit阳性细胞数量比较,差异均有统计学意义(P<0.05);其中过表达GATA-4-BMSCs组心脏区域荧光强度、C-kit阳性细胞数量多于对照1组、对照2组、对照3组、BMSCs组和空载体-BMSCs组(P<0.05),心肌血管数量多于BMSCs组和空载体-BMSCs组。结论过表达GATA-4-BMSCs可以通过增强"归巢"效应、促进心肌梗死局部血管增生、有效动员C-kit阳性细胞修复心肌损伤。
Objective This study explores the mechanism of murine bone marrow mesenchymal stem cells（BMSCs） with over-expressed GATA-4 in repairing myocardial injury.Methods From March 2015 to December 2016,sixty healthy SPF male C57 BL/6 mice aged 4 weeks were selected.BMSCs were isolated and the expression rates of CD29,CD44,CD11 b and SCA-1 were detected by flow cytometry.Over-expressed GATA-4 BMSCs were synthesized by transfecting the murine BMSCs with GATA-4-carrying slow virus vector GV308,subsequently added the gene initiator doxycycline（DOX）.The expression levels of GATA-4 m RNA in the over-expressed GATA-4-BMSCs after transfection 72 h were detected by RT-PCR,and no transfection was used as negative control.The myocardial infarction models of mice were successfully established,BMSCs（n=3）,blank vector-BMSCs（n=3）,over-expressed GATA-4-BMSCs（n=3）500 μl were injected via the caudal vein.And the myocardial infarction untreated group（n=3） and normal mice（n=3） fed with DOX-containing liquid at the same time were selected as control group 1 and control group 2.The heart function improvement was detected through cardiac color Doppler ultrasonography 72 h after providing the intervention measures.Normal mice with normal feeding（n=3） were selected as control group 3.The fluorescence intensity of heart region was detected by in vivo imaging of small animals.The local myocardial blood vessels in myocardial infarction were evaluated by tomato lectin,and the number of local C-kit positive cells in myocardial infarction was detected using immunohistochemistry.Results The expression rate of BMSCs CD29 in mice was 98.0%,the expression rate of CD44 was 100.0%,the expression rate of CD11 b was 0.1% and the expression rate of SCA-1 was 99.5%.The expression level of GATA-4 m RNA in GATA-4-BMSCs group was（78.17±1.32）,which was higher than that in the negative control group（0.57±0.34）（t=2.576,P〈0.05）.The ejection fraction（EF） and central systolic（FS） of each group were compared,and t
出处
《中国全科医学》
CAS
北大核心
2018年第2期167-172,共6页
Chinese General Practice
基金
国家自然科学基金资助项目(81460073)
云南省科技厅-昆明医科大学应用基础研究联合专项(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)
