Adenovirus-mediated vascular endothelial growth factor transfecting cardiomyocyte in vitro

Objective:To construct a recombinant adenovirus vector harboring hVEGF 165 cDNA under the control of ven-tricle-specific myosin light chain-2(mlc-2v)promotor and to detect whether it can specifically transfect cardiomyocyte in vitro. Methods:PDC317was built by removing the native promoter CMV of PDC315.Human vascular endothelial growth factor (hVEGF 165 )cDNA and mlc-2v were cloned into adenovirus shuttle vector PDC317by standard procedure,and the product was identified and transferred to the adenoviral packaging cell HEK293cells mediated by lipofectamine.The desired Ad vec-tors were purified by density gradient ultracentrifuge and titrated in293cells after identified.The neonatal rat cardiomy-ocytes,skeletal muscle cells and smooth muscle cells were transfected with Ad-mlc-hVEGF 165 and Ad-hVEGF 165 in vitro,re-spectively.The expression of VEGF 165 proteins was assessed by ELISA and Western blot.Results:The segment length of PDC-mlc-VEGF and the correct clones containing target gene in right direction was identical.The titer of adenovirus reached 2.8×10 9 pfu/ml after density gradient ultracentrifuge.ELISA and Western blot showed that all the3cells secreted VEGF protein in Ad-hVEGF 165 group,and only cardiomyocyte secreted VEGF 165 proteins in Ad-mlc-hVEGF 165 group.VEGF secre-tion in Ad-mlc-hVEGF 165 group was less than that in Ad-hVEGF 165 group.Conclusion:The recombinant adenoviral vector Ad- mlc-hVEGF 165 has been successfully established,which can make the neonate cardiomyocyte secret VEGF 165 in vitro,provid-ing a promising tool for gene targeting therapy of myocardial ischemia.