【摘要】 目的观察同种异体骨髓间充质干细胞(MSCs)联合血管内皮生长因子(VEGF)基因移植对急性心肌梗死(AMI)小型猪心肌细胞凋亡及凋亡相关蛋白Bcl-2、Bax和Caspase-3表达的影响,并探讨其可能机制。方法体外分离、培养骨髓MSCs,腺病毒介导的VEGF基因转染骨髓MSCs。用球囊堵闭法建立猪AMI模型。1周后,随机将其分为4组(每组4只),组Ⅰ:转染VEGF腺病毒的MSCs移植;组Ⅱ:单纯MSCs移植;组Ⅲ:单纯VEGF移植;组Ⅳ:磷酸盐缓冲液对照组。4周后超声心动图检测心功能,采用DNA缺口末端标记法(TUNEL)检测心肌细胞凋亡,测定心肌细胞凋亡指数(AI);免疫组化法检测Bcl-2、Bax、Caspase-3蛋白在心肌细胞中表达水平。结果 (1)超声心动图检查发现:组Ⅰ、Ⅱ、Ⅲ反映心功能的射血分数(EF)、左室短轴缩短率(FS)值较干预前均有明显增加(P<0.01或P<0.05),组Ⅰ与其他3组比较心功能改善最明显(P均<0.01),组Ⅳ干预前后心功能无明显变化(P>0.05)。(2)TUNEL结果显示:与其他3组比较,干预后组Ⅰ心肌细胞AI最低(P均<0.01),Bax、Caspase-3的表达最低(P均<0.05),Bcl-2的表达最高(P均<0.05)。结论骨髓MSCs联合VEGF基因移植猪AMI区可促进血管再生,改善心功能,减少心肌细胞凋亡,抑制Bax、Caspase-3的表达,促进Bcl-2的表达。更多还原

【Abstract】 Objective To observe the impacts of bone marrow mesenchymal stem cells(MSCs) combined with vascular endothelial growth factor(VEGF) gene transplantation on cardiomyocyte apoptosis and expressions of apoptosis-association protein Bcl-2,Bax and Caspase-3 and explore the potential mechanisms in a porcine acute myocardial infarction(AMI) model.Methods The bone marrow MSCs were isolated and cultured in vitro and transferred with adenovirus-mediated VEGF(Ad.VEGF) gene.The porcine AMI model was established by balloon closure method.The minipigs were randomly divided into four groups(n = 4 each) for different aim after one week:groupⅠ(intracoronary infusion of 100 μl auto-MSCs transferred with Ad.VEGF gene),groupⅡ(intracoronary infusion of 100 μl auto-MSCs non-transferred with Ad.VEGF gene),group Ⅲ(intracoronary infusion of 50 μl auto-VEGF plasmid) and group Ⅳ(intracoronary infusion of 100 μl phosphate buffer solution as a control).Four weeks later,the heart function was determined by echocardiography in vivo,then the minipigs were killed and the hearts were dissected for measuring cardiomyocyte apoptosis and the expressions of apoptosis-association protein in vitro.The cardiomyocyte apoptosios was measured by TdT-mediated dUTP nick end labeling(TUNEL) method,and the cardiomyocyte apoptosis index(AI) was calculated.The expression levels of Bcl-2,Bax and Caspase-3 protein in cardiomyocyte were measured by immunohistochemical method.Results(1) Echocardiogram showed that ejection fraction(EF) and left ventricular fractional shortening rate(FS) were significantly improved compared with before intervention in groups Ⅰ,Ⅱ and Ⅲ(P < 0.01 or P < 0.05),and the improvement of heart function in group Ⅰwas the most obvious(P < 0.01).The heart function remained unchanged before and after intervention in group Ⅳ(P > 0.05).(2) The results of TUNEL showed that the AI value and the expression levels of Bax and Caspase-3 proteins in cardiomyocytes were all the lowest(P < 0.01 or P < 0.05),and the expression level of Bcl-2 protein was the highest(P < 0.05) in group Ⅰcompared with the other groups.Conclusions Bone marrow MSCs combined with VEGF gene transplantation to porcine AMI region has the effects of promoting angiogenesis,decreasing cardiomyocyte apoptosis,inhibiting expressions of Bax and Caspase-3,enhancing the expressions of Bcl-2 and thus improving heart function.更多还原