携hVEGF_(165)基因的重组腺病毒载体的构建及鉴定

Construction and Identification of Recom binant Adenovirus Vector Containing the hVEGF_(165) Gene

目的 构建携带人血管内皮生长因子 16 5 (hVEGF165)基因的重组腺病毒载体。方法 将hVEGF165cDNA亚克隆到腺病毒中间载体pACCMV·pLpA ,再与pJM17共转染人胚肾 2 93细胞 ,获得载hVEGF165基因的复制缺陷型重组腺病毒 ,感染体外培养的兔主动脉血管平滑肌细胞 (VSMC) ,通过RT PCR和Westernblot检测VEGF表达情况 ,并观察表达产物VEGF对人脐静脉内皮细胞 (HUVEC)增殖的影响。结果 重组腺病毒感染VSMC 4 8h后有VEGFmRNA转录及蛋白质的表达 ,并呈剂量依赖性地促进HUVEC增殖。结论 构建的重组腺病毒载体在VSMC中能够有效表达目的基因 ,且能有效促进HUVEC增殖 ,为hVEGF165基因的实际应用奠定了基础。

Objective To construct the recombinant adenovirus vector contai ning the cDNA for human vascular endothelial growth factor-165 (hVEGF 165) . Methods The cDNA for hVEGF 165 was subcloned into pACCMV· pLpA and subsequently this recombinant pACCMV·hVEGF was co-transfected into 29 3 cells together with pJM17 to obtain the replication-deficient recombinant ade novirus containing hVEGF gene-AdCMV·hVEGF. The VEGF gene expression was detect ed by RT-PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSM C) infected with AdCMV·hVEGF. Cultured human umbilical vein endothelial cells ( HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV·hVEGF to observe the effect of VEGF on proliferation of HU VEC. Results48 h after infection with AdCMV·hVEGF, VSMC demonstrated VEGF expression and the expressed VEGF could stimulate the proliferation of HUV EC in vitro. Conclusion Successfully constructed AdCMV·hVEGF 165 could express biological active VEGF in infected VSMC, and stimulate proliferation of HUVEC.