重组腺相关病毒介导人血管内皮生长因子165基因在培养大鼠心肌细胞中的表达

Expression of Human Vascular Endothelial Growth Factor 165 Gene Mediated by Recombinant Adeno-associated Viral Vector2 in Rat Cardiomyocytes in vitro

目的探讨2型腺相关病毒载体介导的人血管内皮生长因子165基因在离体心肌细胞中的表达及表达产物的生物活性。方法构建携带人血管内皮生长因子的2型腺相关病毒载体(rAAV-2/hVEGF165),感染大鼠原代心肌细胞,采用逆转录聚合酶链反应、免疫印迹法和酶联免疫吸附测定等方法检测心肌细胞人血管内皮生长因子165的表达,应用MTT法检测转染后细胞上清液中血管内皮生长因子165的生物学活性。结果逆转录聚合酶链反应检测结果发现感染的心肌细胞可表达人血管内皮生长因子165 mRNA;免疫印迹法检测结果表明感染的心肌细胞中有人血管内皮生长因子165,酶联免疫吸附测定法检测到血管内皮生长因子蛋白分泌水平为546.5±15.6pg/L,未转染组未检测到血管内皮生长因子蛋白的分泌;还发现感染的心肌细胞上清具有明显的促人内皮细胞增殖作用。结论2型腺相关病毒载体介导的人血管内皮生长因子165可有效转染大鼠心肌细胞,且表达产物具有促内皮细胞增殖作用。

Aim To investigate vascular endothelial growth factor （VEGF） gene expression and bioactivity in rat cardiomyocytes transfected by a recombinant adeno-associated viral vector 2. Methods Recombinant adeno-associated viral vector 2 encoding the human VEGF 165 were constructed and confected in intro to the neonate rat ventricular myocytes. RT-PCR, Western blot and ELISA were used to detect the expression of VEGF gene, and MTF to detect the biological activity of the conditioned medium after transfection. Results The presence of VEGF165 mRNA in transfected cells was demonstrated by RTPCR, the expression of VEGF165 protein was confirmed by western blot. The level of VEGF165 protein expressed by transfected cells was higher than that by control accroding to an enzyme-hnked immtmosorbent assay. The conditioned medium after transfection could stimulate the proliferation of endothelial cells. Conclusion Rat cardiomyocytes transfected by Recombinant adenoassociated viral vector 2 containing the human vascular endothelial growth factor 165 cDNA （ rAAV-2/hVEGF165 ） could express VEGF165 with highly biologial activity, which might be useful for gene therapy of myocardial ischemia.