腺病毒介导的血管内皮生长因子体外转染心肌细胞的研究

Adenovirus-mediated vascular endothelial growth factor transfecting cardiomyocyte in vitro

目的:构建携带人血管内皮生长因子(VEGF)基因的重组腺病毒载体,并转染体外培养的心肌细胞,检测VEGF的表达。万法:将人源性的VEGF165 cDNA正向插入到腺病毒载体PDC315,构建重组质粒,通过脂质体共转染293细胞,经同源重组获得携带人VEGF165基因的重组腺病毒,通过PCR扩增法鉴定所构建的腺病毒,扩增并测定滴度后,体外转染培养的心肌细胞,利用ELISA、Western印迹分析等方法检测VEGF在心肌细胞中的表达。结果:人VEGF165cDNA成功地正向插入到PDC315载体中,以重组病毒基因组DNA为模板,同时扩增出了610 bp的VEGF165cDNA基因片段,证实了所构建病毒的正确性,病毒滴度为2.8×108pfu／ml,Ad VEGF165体外转染心肌细胞3 d后,在培养细胞的上清液及细胞内检测到了VEGF的表达。结论:成功构建了表达人VEGF165基因的腺病毒载体,体外转染心肌细胞后能够满意表达VEGF,为基因治疗心肌缺血奠定基础。

Objective:To establish a recombinant adenovirus vector harboring human vascular endothelial growth factor (hVEGF165) cDNA and to study VEGF165 expression when the recombinant vectors are used to transfect neonatal rat car-diomyocytes in vitro. Methods :hVEGF165 cDNA were cloned into adenovirus shuttle vector PDC315 by standard procedures. The recombinant adenoviral plasmid was identified and then transferred to the adenoviral packaging HEK 293 cell by lipofec-tamine mediated gene transfer method. PCR amplification was used to identify the constructed adenovirus and the virus titer was determined after amlification. The neonatal rat cardiomyocyte were transfected with the adenovirus in vitro and the expression of VEGF165 proteins was detected using ELISA and Western blot. Results: The recombinant adeno-hVEGF165 was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. ELISA and Western blot showed that the cardiomyocyte secreted VEGF165 protein; furthermore, the concentration reach the highest on the third day. Conclusion: The recombinant adenoviral vector vith hVEGF165 cDNA is successfully established. The transfected cardiomyocyte can express VEGF165 in -vitro, which provides a basis for adenovirus mediated VEGF gene therapy for ischemic heart disease.