腺病毒介导血管内皮生长因子基因转染大鼠骨髓间充质干细胞移植梗死心肌的血管再生

Angiogenesis of infarct myocardium by the transplantation of bone marrow mesenchymal stem cells transfected by vascular endothelial growth factor in rats

背景：骨髓间充质干细胞的存活率与良好的血液供应关系密切，在移植早期其自身所分泌产生的促血管生长因子不足以刺激血管新生而容易导致移植的细胞死亡。目的：观察骨髓间充质干细胞移植联合血管内皮生长因子基因转染对大鼠梗死心肌组织修复重建、血管再生的影响。设计、时间及地点：随机分组设计的细胞基因工程实验，于2006-01／2007-12在中山大学第二附属医院医学研究中心完成。材料：选取具有相同遗传背景的雄性SD大鼠40只建立急性心肌梗死模型，6~8周龄．体质量250～300g。方法：体外分离、培养、纯化SD大鼠骨髓间充质干细胞，以BrdU标记骨髓间充质干细胞。腺病毒介导血管内皮生长因子基因转染骨髓间充质干细胞。将造模后大鼠随机分为4组．每组10只：基因转染骨髓间充质干细胞组，梗死心肌内注射体外转染腺病毒介导血管内皮生长因子的骨髓间充质干细胞；骨髓间充质干细胞组，梗死心肌内注射骨髓间充质干细胞；基因转染组，注射腺病毒介导的血管内皮生长因子；对照组，注射无血清IMDN培养液。主要观察指标：移植4周后观察移植细胞分化和新生血管的形成，并通过超声多普勒检测心功能变化。结果：细胞移植4周后，基因转染骨髓间充质干细胞组、骨髓间充质干细胞组移植区移植细胞表现为心肌肌钙蛋白T染色阳性，细胞呈肌样细胞形态，并且两组心功能都较移植前有明显改善，基因转染骨髓间充质干细胞组改善程度优于骨髓间充质干细胞组。部分BrdU染色阳性的细胞可以分化成为内皮细胞，参与构成了梗死区域新生毛细血管，相对于单纯细胞移植与细胞因子移植，细胞移植结合血管内皮生长因子基因转染促进毛细血管新生更明显。结论：骨髓间充质干细胞移植联合血管内皮生长因子基因转染可以通过促进心肌再生和新生血管的形成来重建缺血心肌，明显改善心功能。

BACKGROUND： The livability of bone marrow mesenchymal stem cells （MSCs） nearly involves in the well blood supply. The vascular growth factor excreted by MSCs in the early period of transplantation could not stimulate the angiogenesis, thus conducing the death of transplanted cells. OBJECTIVE： To investigate the effects of MSCs transplantation combining vascular endothelial growth factor （VEGF） gene therapy on the tissue repair and angiogenesis in rats with myocardial infarction. DESIGN, TIME AND SETTING： Randomized group experiments of cell genetic engineering were carried out in the Medical Experimental Center of the Second Affiliated Hospital of Sun Yat-sen University （Guangzhou, Guangdong, China） from January 2006 to December 2007. MATERIALS. In the same genetic background, 40 male SD rats were used to establish the acute myocardial infarction models, aged 6 8 weeks old and weighing 250 300 g. METHODS： Rat MSCs were isolated, cultured and purified in vitro, then labeled with BrdU and transfected by adenovirus VEGF （Ad.VEGF） gene. Four weeks after creating the myocardial infarction, forty rat models were randomly divided into four groups （n=10）： MSCs/Ad.VEGF group, MSCs group, Ad.VEGF group and control group. The former three groups were treated with corresponding injections, and control group was injected with serum-free IMDM. MAIN OUTCOME MEASURES- MSCs differentiation and angiogenesis were observed 4 weeks after transplantation. Doppler echocardiography was performed to detect the changes on heart function. RESULTS： In the MSCs/Ad.VEGF and MSCs groups, the MSCs were positively stained with cardiac troponin T antibody at 4 weeks after transplantation, and the cells were shaped as myoid cells. The heart function was obviously improved after transplantation, and the improvement was the best in the MSCs/Ad.VEGF group. Some BrdU-stained MSCs differentiated into the endothelial cells and incorporated into the coronary capillaries in the infarcted region. Compared to the MSCs or VEGF transplantation alone, the MSCs/Ad.VEGF transplantation could facilitate the angiogenesis. CONCLUSION： MSCs transplantation combining VEGF gene therapy can obviously promote the myocardial regeneration, enhance the angiogenesis and improve the heart function in the infarct myocardium.