关键词: 血管内皮细胞生长因子, 真核表达载体, NIH3T3细胞系, 基因表达

Abstract: Objective To study human vascular endothelial growth factor(hVEGF) gene transfer and expression in NIH3T3 fibroblast cell line,and establish the experimental foundation of angiogenesis tissue engineering organization and the treatment of ischemic disorders.Methods pcDNA/V eukaryotic expression vector and pcDNA3.1(+) were transfected into NIH3T3 cells mediated by liposome.The transfected cells were grown in DMEM medium containing G418 for 72 hours,and the clones of cells were selected and continued to grow in G418 medium.The cells stably expressing hVEGFwere selected under the pressure of G418.The selected cells were grown in DMEMmedium containing G418 for 72 hours,and then the supernatant was collected.The expressed hVEGFprotein was analysed by using Western blot and immunohistochemical methods.Results Western blotting showed that hVEGFof 22000 was detected in culture medium of NIH3T3 cells transfected with pcDNA/V.Immunohistochemistry demonstrated that the NIH3T3 cells transfected with pcDNA/V showed positive reaction to VEGFin cytoplasm,whereas faint reaction was observed in non-transfected cells and pcDNA3.1(+)-transfected cells.Conclusion Human VEGFgene was successfully transfected and its protein could be expressed in NIH3T3 cells.

Key words: vascular endothelial growth factor, eukaryotic expression vector, NIH3T3 cell line, gene expression