血管内皮生长因子基因转染促进骨髓间质干细胞增殖的实验

目的：以基因转染为平台，利用脂质体将血管内皮生长因子基因转入骨髓间质干细胞，使其可以在作为种子细胞的同时。作为基因治疗的载体将血管内皮生长因子基因导人缺血组织，从而为探索一种更加有效的缺血性心脏病治疗方法提供实验室基础。 方法：实验于2004—07／2005-03在解放军第二军医大学医学遗传实验室及上海交通大学附属第一人民医院心内科实验室完成。①分离、培养、鉴定大鼠骨髓间质干细胞。②试验组用构建好的PcDNA3．1-VEGF进行基因转染，阳性对照组用PcDNA3．1空质粒进行转染，阴性对照组只加入培养液。③收集各组第1，3，5，7，9，11天的上清液，用ELISA法测定培养上清液中血管内皮生长因子的浓度；提取阳性克隆的细胞蛋白；进行Western blot分析；用四甲基偶氮唑盐法检测转染后血管内皮生长因子的表达对骨髓间质于细胞增殖的影响。 结果：①免疫组织化学示所培养细胞CD106^＋，CD34^-。②ELISA方法示质粒转染细胞后第2天，上清液中即可出现血管内皮生长因子浓度的增高，第5天时达到高峰，以后逐渐降低，10d后趋于稳定，但其浓度仍高于对照组。③Westem blot示转染组血管内皮生长因子蛋白浓度明显高于空质粒转染组和未转染组。④四甲基偶氯唑盐示转染后血管内皮生长因子的表达可以促进骨髓间质干细胞的增殖。 结论：血管内皮生长因子成功转染骨髓间质干细胞，血管内皮生长因子的表达促进了骨髓间质干细胞的增殖，从而可以将促血管生长因子治疗和干细胞移植有效的结合起来。

Transfection of vascular endothelial growth factor gene in improving the proliferation of bone mesenchymal stem cells

AIM： To provide further foundation for the therapy for ischemic heart diseases by transferred bone mesenchymal cells with the recombinant plasmid coding vascular endothelial growth factor by lipofectamine.
METHODS： The experiment was conducted at the laboratory of Medical Genetics, Second Military Medical University of Chinese PLA and the laboratory of Department of Cardiology, Shanghai First People＇s Hospital of Shanghai Jiao Tong University from July 2004 to March 2005. （1）Bone mesenchymal stem cells were isolated from rat and directly cultivated and expanded in vitro. （2）Bone mesenchymal cells were divided into 3 groups： PcDNA3.1-VEGF transfected group; PcDNA3.1 transfected group and untransfected group. （3）The supernatant of each group was collected at day 1, 3, 5, 7, 9, 11 to detect the subsequent protein expression of vascular endothelial growth factor by ELISA method. Western blotting was applied to assay the expression of transgenic bone mesenchymal stem cells cultured in L-DMEM containing G418. MTT method was applied to assay the mitogenic effect of expression of vascular endothelial growth factor on bone mesenchymal stem cells.
RESULTS： （1）immunohistochemistry showed CD106 was positive and CD34 was negative. （2）ELISA suggested that one day after the beginning of transfection, the concentration of vascular endothelial growth factor in the supernatant of medium was significantly increased, and reached its peak at the fifth day; then it gradually decreased and kept stable ten days later, but still higher than that of the control group. （3）Western blot showed that the vascular endothelial growth factor protein of the PcDNA3.1-VEGF transfected group was higher than that in PcDNA3.1 transfected group and untransfected group. （4） MTT showed that the expression of vascular endothelial growth factor could improve the proliferation of bone mesenchymal stern cells.
CONCLUSION： The plasmid vector coding vascular endothelial growth factor gene is successfully transfected