摘要
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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2007年第4期408-411,共4页
Academic Journal of Second Military Medical University
关键词
成纤维细胞生长因子2
腺病毒科
基因表达
脐静脉
内皮
血管
basic fibroblast growth factor 2
adenoviridae
gene expression
umbilical veins
endothelium,vascular