血管内皮生长因子表达载体的构建及在内皮祖细胞中的表达

背景：血管内皮生长因子(vascular endothelial growth factor, VEGF)具有很强的促进血管生成作用，但构建其真核表达质粒是否可转染血管内皮祖细胞并完整表达还不清楚。 目的：构建VEGF表达载体,并观察其在内皮祖细胞中的表达情况。 设计、时间及地点：观察性试验，于2007-12/2008-03在上海交通大学医学院附属新华医院科研中心实验室完成。 材料：质粒PDC315-VEGF165自备，质粒pIRES2-EGFP由美国国立卫生研究院Stanko Stojilkovic教授惠赠。 方法：运用DNA重组技术构建VEGF表达载体pIRES2-EGFP-VEGF，应用脂质体包裹的方法将载体转染猪外周血血管内皮祖细胞。 主要观察指标：采用荧光显微镜观察VEGF在内皮祖细胞内的表达，反转录-聚合酶链反应法检测VEGFmRNA表达水平， ELISA检测VEGF165蛋白表达情况。 结果：成功构建VEGF真核表达载体pIRES2-EGFP-VEGF。转染重组质粒pIRES2-EGFP-VEGF后,在内皮祖细胞内有VEGF的表达，VEGFmRNA和VEGF165 蛋白表达水平均明显增加。 结论：VEGF 表达载体转染猪外周血血管内皮祖细胞后能有效增加VEGF基因的表达，能够获得较高水平的VEGF蛋白。

Expression of vascular endothelial growth factor in endothelial progenitor cells following its expression vector construction

BACKGROUND: Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. However, whether the construction of eukaryotic expression vector can transfect and express in endothelial progenitor cells is unclear. OBJECTIVE: To construct a eukaryotic expression vector, further more, to investigate its expression in endothelial progenitor cells. DESIGN, TIME AND SETTING: The sample observation experiment was conducted in the Center Laboratory of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine from December 2007 to March 2008. MATERIALS: The vector PDC315-VEGF165 was self-made in the laboratory. The vector pIRES2-EGFP was provided by Stanko Stojilkovic, a professor from American National Institutes of Health. MEHODS: pIRES2-EGFP-VEGF expression vector was constructed by DNA recombinant technique and was introduced into pig endothelial progenitor cells by liposome-encapsulated transfection. MAIN OUTCOME MEASURES: VEGF165 expression in endothelial progenitor cells, level of VEGF mRNA and protein VEGF165 expression were identified by fluorescence microscope, RT-PCR and ELISA, respectively. RESULTS: pIRES2-EGFP-VEGF expression vector was successfully constructed. When transfection recombinant plasmid pIRES2-EGFP-VEGF, VEGF expression could be found in endothelial progenitor cell, VEGF mRNA and protein VEGF165 levels were obvious increased. CONCLUSION: Transfection VEGF expression vector with pig endothelial progenitor cells can effectively enhance the expression of VEGF gene, can obtain high level of VEGF protein.