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目的探讨重组腺病毒介导的血管生成素-1(Ang-1)基因转染兔外周血血管内皮祖细胞(EPCs)的方法及效果。方法从兔外周血中体外分离、扩增EPCs,采用免疫组织化学和荧光化学法鉴定EPCs,设EPCs细胞对照组(EPCs)、EPCs+Ad空病毒载体对照组(EPCs+Ad)及EPCs+Ad-Ang-1组(EPCs+Ad-Ang-1)。以携带Ang-1基因的重组腺病毒转染EPCs后,用荧光显微镜下观察转染效果,RT-PCR鉴定细胞内Ang-1基因在EPCs中的转录;ELISA法检测细胞培养上清中的Ang-1蛋白表达。结果 EPCs+Ad-Ang-1组镜下观察到大部分细胞呈现绿色荧光;RT-PCR鉴定证实EPCs细胞内的Ang-1转录;ELISA检测证实EPCs+Ad-Ang-1组细胞上清液中Ang-1的浓度高于EPCs组和EPCs+Ad组(均P<0.05)。结论腺病毒介导的Ang-1基因转染外周血EPCs可行。
Objective To investigate the method and effect of recombinant adenovirus-mediated transfection of angiopoietin-1 (Ang-1) gene into rabbit peripheral blood endothelial progenitor cells (EPCs). Methods EPCs were isolated and expanded from peripheral blood of rabbits in vitro. EPCs were identified by immunohistochemistry and fluorescent staining. EPCs, EPCs + Ad, EPCs + Ad, -Ang-1 group (EPCs + Ad-Ang-1). Transfection of EPCs with recombinant adenovirus carrying Ang-1 gene was carried out. The transfection efficiency was observed under a fluorescence microscope. The transcription of Ang-1 gene in EPCs was identified by RT-PCR. The levels of Ang-1 -1 protein expression. Results EPCs + Ad-Ang-1 group showed green fluorescence in most of the cells under microscope; Ang-1 transcription in EPCs was confirmed by RT-PCR and ELISA in the supernatants of EPCs + Ad-Ang- The concentration of Ang-1 was higher than that of EPCs and EPCs + Ad groups (all P <0.05). Conclusion Adenovirus-mediated transfection of peripheral blood EPCs with Ang-1 gene is feasible.