人碱性成纤维细胞生长因子腺病毒表达载体的构建及其在人脐静脉内皮细胞中的表达

Construction of basic fibrobiast growth factor adenovinis vector and its expression in human umbilical vein endothelial cells

目的:构建人碱性成纤维细胞生长因子(basic fibroblast cell growth factor,bFGF)腺病毒载体,并观察其在体外血管内皮细胞中的表达。方法:采用同源重组的方法,构建重组腺病毒Ad5-bFGF。携有绿色荧光蛋白(green fluorescence pro- tein,GFP)的Ad5腺病毒转染人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),检测最佳转染复数。Ad5- bFGF体外转染HUVEC,免疫细胞化学法、Western印迹法检测bFGF蛋白的表达。结果:Ad5转染HUVEC的最佳转染复数为200,转染率为90％。免疫细胞化学法和Western即迹结果显示bFGF基因以Ad5为载体可以在HUVEC的胞质和胞核表达．并且表达量较未转染的HUVEC明显增加。结论:成功构建了携带bFGF基因的腺病毒载体,体外可以成功表达,为bFGF基因治疗及肿瘤发病机制的研究奠定了基础。

Objective：To construct an adenovirus vector harboring human basic fibroblast growth factor （bFGF） cDNA and investigate the expression of bFGF in human umbilical vein endothelial cells （HUVEC） in vitro. Methods： The adenovirus expression vector Ad5-bFGF was constructed by homologous recombination technique. The best value of MOI was tested by transfecting human umbilical vein endothelial cells （HUVEC） with Ad5-GFP. Ad5-bFGF was used to transfect HUVEC at the obtained value of MOI and the expression of bFGF protein was detected by immunocytochemistry method and Western blotting, Results： The best value of MOI for adenovirus 5 to transfect HUVEC was 200 and the transfection rate was 90%. Immunocytochemistry method and Western blotting showed that bFGF was expressed in HUVEC after transfection with Ad5- bFGF and the expression was significantly higher than that in untransfected HUVEC （P〈 0. 05）. Conclusion： We have successfully constructed a recombined adenovirus vector Ad5-bFGF which can be expressed in vitro in HUVEC, paving a way for application of bFGF in gene therapy and study of tumorigenesis.