Bcl-2改善老年人骨髓间充质干细胞抗缺氧损伤能力的研究

The anti-hypoxic injury ability of bone marrow mesenchymal stem cells transfected by bcl-2

目的:通过转染Bcl-2基因增强老年人骨髓间充质干细胞(hMSCs)的Bcl-2表达,尝试改善老年hMSCs在缺氧环境下的抗凋亡、抗损伤能力,为基因联合细胞移植改善老年hMSCs自体移植治疗缺血性心脏病的提供依据。方法:分离培养老年hMSCs并进行鉴定。构建Bcl-2基因质粒并对老年人hMSCs进行Bcl-2基因的转染。应用CO_2和N_2的混合气体建立稳定的缺氧培养环境模拟心肌梗死时的缺氧环境,分别对老年hMSCs及Bcl-2转染后老年hMSCs进行缺氧培养,比较各组细胞的细胞凋亡及损伤情况。通过冠状动脉结扎建立稳定的大鼠心肌梗死模型,分别应用老年hMSCs及Bcl-2转染后的老年hMSCs进行细胞移植,比较各组细胞在心肌梗死区域缺氧环境下的存活情况,同时应用超声心动图对比两组细胞移植后心肌梗死大鼠心脏功能的改善情况。结果:转染Bcl-2的老年hMSCs能够稳定表达Bcl-2的基因及蛋白。转染Bcl-2后的老年hMSCs在缺氧环境下较未转染的老年细胞其凋亡数量明显下降、生存率明显提高,表达的Bcl-2基因及蛋白也多于未转染的老年细胞。各组细胞移植至大鼠梗死心脏后可见转染Bcl-2的老年hMSCs组的细胞存活数量明显高于未转染组,梗死心脏的Bcl-2的基因含量和蛋白表达量也高于未转染老年细胞移植组,心肌梗死大鼠在移植了转染Bcl-2的老年hMSCs组后其心脏的射血分数和左心室短轴缩短率好于未行细胞移植组和移植未转染细胞组。结论:增加老年hMSCs中Bcl-2基因的含量能够增加其Bcl-2蛋白表达量、增强其在缺氧环境中的抗凋亡能力。应用Bcl-2转染的方法能够增强老年hMSCs移植治疗心肌梗死时的移植后细胞存活能力、提高梗死区域的Bcl-2蛋白表达量并增强了老年hMSCs移植改善心肌梗死后心脏功能的效果。

Object:The aim of this study was to enhance the expression of Bcl-2 in human mesenchymal stem cells(hMSCs) by transfecting Bcl-2 gene, and to improve the anti-apoptotic and anti-injury ability of hMSCs in hypoxic environment. Furthermore,we try to provide data for the improvement of hMSCs autologous transplantation for elderly patients with ischemic heart disease. Methods: Bone marrow mesenchymal stem cells were obtained from young and old people. A stable hypoxic culture environment was established by using a mixture of CO2 and N2, and a stable rat model of myocardial infarction was established by coronary artery ligation. The Bcl-2 gene was transfected into the aged bone marrow mesenchymal stem cells by constructing a plasmid that can stably carry the Bcl-2 gene as a transfection vector. Bone marrow mesenchymal stem cells(BMSCs) and Bcl-2 transfected human bone marrow mesenchymal stem cells were cultured in hypoxia. The apoptosis and injury of the cells in each group were detected and compared. Each group of cells was used for cell transplantation in a rat model of myocardial infarction to observe and compare the survival rate of each group of cells in the hypoxic environment of the myocardial infarction region. At the same time, echocardiography was used to compare the improvement of cardiac function in rats with myocardial infarction after transplantation. Results: Bone-derived mesenchymal stem cells transfected with Bcl-2 can stably express Bcl-2 genes and proteins. BMSCs transfected with Bcl-2 significantly decreased the number of apoptotic cells, increased the survival rate, and made more Bcl-2 genes and proteins expressed than the un-transfected aged cells in hypoxia. After transplanting both groups of cells into the infarcted heart of rats, the number of surviving cells in the Bcl-2-bearing old bone marrow mesenchymal stem cells group was significantly higher than that in the non-transfected group, and the Bcl-2 gene content and protein expression in the infarcted hearts is also higher than the un-transfected aged cell transplantation group. The ejection fraction and the left ventricular fractional shortening were better than the un-transplanted group and the un-transfected group. Conclusions: Increasing the content of Bcl-2 gene in bone marrow mesenchymal cells of the elderly can increase the expression of Bcl-2 protein and enhance its anti-apoptosis ability in hypoxic environment. The method of Bcl-2 transfection can enhance the post-transplant cell viability of aged bone marrow mesenchymal stem cells transplantation and increase the expression of Bcl-2 protein in the infarcted region, thus confirming that the transfected Bcl-2 can enhance the effect of elderly hMSCs on improving cardiac function after myocardial infarction by transplantation.