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目的探讨人血管内皮细胞生长因子165(hVEGF165)基因转染对人外周血内皮祖细胞(EPCs)的影响。方法体外分离、培养、鉴定人外周血EPCs。实验分脂质体介导pcDNA3.1-hVEGF165质粒转染组,pcDNA3.1空质粒转染组,空白对照组。ELISA法和硝酸还原酶法分别测定各组上清液中VEGF和一氧化氮(NO)的含量;噻唑蓝(MTT)检测它们对EPCs增殖的影响。结果FITC-UEA-I和DiI-ac-LDL双染色阳性细胞为正在分化的EPCs,脂质体介导pcDNA3.1-hVEGF165质粒转染组EPCs培养基上清液中VEGF和NO表达量明显高于其他两组(P<0.01);VEGF质粒转染对EPCs的增殖无明显影响。结论人外周血EPCs可以成功转染hVEGF165基因,同时能表达一定浓度的VEGF蛋白,并可促进NO的分泌,而对EPCs的活性无明显影响。该研究为进一步研究VEGF165基因和EPCs结合治疗缺血性疾病提供了实验依据。
Objective To investigate the effect of human vascular endothelial growth factor 165 (hVEGF165) gene transfection on human peripheral blood endothelial progenitor cells (EPCs). Methods The EPCs in human peripheral blood were isolated, cultured and identified in vitro. The experiment was divided into liposome-mediated pcDNA3.1-hVEGF165 plasmid transfection group, pcDNA3.1 empty plasmid transfection group, blank control group. ELISA and nitrate reductase were used to determine the contents of VEGF and nitric oxide (NO) in the supernatants of each group. The effects of them on the proliferation of EPCs were detected by MTT assay. Results The double positive cells of FITC-UEA-I and DiI-ac-LDL were differentiated EPCs. The expression of VEGF and NO in supernatant of EPCs transfected with pcDNA3.1-hVEGF165 plasmid was significantly higher In the other two groups (P <0.01), VEGF plasmid transfection had no significant effect on EPCs proliferation. Conclusion Human peripheral blood EPCs can successfully transfect hVEGF165 gene, express VEGF protein at a certain concentration and promote the secretion of NO, but have no obvious effect on the activity of EPCs. This study provides the experimental evidence for further study of the combination of VEGF165 gene and EPCs in the treatment of ischemic diseases.