腺病毒介导的血管内皮生长因子体外靶向性转染心肌细胞

Adenovirus mediated vascular endothelial growth factor transfecting cardiomyocyte in vitro

目的 :构建以鼠心肌轻链蛋白基因 (mlc- 2 v)为启动子、携带人血管内皮生长因子 h VEGF1 6 5基因的重组腺病毒载体 ,检测该重组腺病毒载体对心肌细胞转染的靶向性。 方法 :酶切腺病毒载体 PDC31 5自身启动子 CMV,构建腺病毒载体PDC31 7,分别接入 h VEGF1 6 5、mlc- 2 v基因 ,构建腺病毒载体 PDC- mlc- h VEGF1 6 5。鉴定正确后 ,将 PDC- mlc- h VEGF1 6 5与 L ipo-fectam ine共转染至 2 93细胞 ,经同源重组获得以 m lc- 2 v为启动子、携带 h VEGF1 6 5基因的重组腺病毒 Ad- m lc- h VEGF1 6 5,同步构建无靶向性的重组腺病毒 Ad- h VEGF1 6 5。扩增并测定滴度后 ,Ad- h VEGF1 6 5、Ad- mlc- h VEGF1 6 5分别转染体外培养的心肌细胞、骨骼肌细胞及平滑肌细胞 ,利用 EL ISA、Western印迹分析等方法检测 Ad- mlc- h VEGF1 6 5转染心肌细胞的靶向性。 结果 :构建的病毒正确 ,病毒滴度为 2 .8× 1 0 9pfu/ ml。转染 3d后 ,Ad- h VEGF1 6 5组在各培养细胞的上清液及细胞内均检测到 VEGF的表达 ,而 Ad- mlc- h VEGF1 6 5组仅在心肌细胞中有 VEGF的表达 ,且 Ad- m lc- h VEGF1 6 5组心肌细胞分泌的 VEGF要少于 Ad-h VEGF1 6 5组。结论 :成功构建了以 mlc- 2 v为启动子、携带人 VEGF基因的重组腺病毒 Ad- m lc-

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.