人VEGF165基因转染人外周血内皮祖细胞的实验研究

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,窄白对照组.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.

Experimental study of human VEGF165 gene transfection into endothelial progenitor cells of human peripheral blood

Objective:To investigate the feasibility of human VEGF165 gene transfection into endothelial progenitor cells (EPCs) derived from human peripheral blood,and the influence of transfusion on EPCs.
Methods:EPCs were isolated from human peripheral blood, cultured in vitro，identified by FITC-UEA-I and Dil-ac-LDL. EPCs was transfected with pcDNA3.1-hVEGF165 by liposomal，with  pcDNA3.1 by liposomal and EPCs without transfection. After transfection, the expression of VEGF and NO in culture supernatants were detected by using ELISA and nitrate reductase method.The proliferation of EPCs after gene transfection was detected by MTT methods.
Results:Cells that expressed double FITC-UEA-I and Dil-ac-LDL fluorescence were EPCs.The expression of VEGF and NO in the medium of EPCs transfected with pcDNA 3.1-hVEGF165 was obviously more than that in the other two transfected groups(P＜0.01). No influence of EPCs proliferation could be found after transfection.
Conclusions:EPCs from human peripheral blood can be successfully transfected with VEGF165 gene, and can express a certain concentration of VEGF protein and promote the secretion of NO，but no influence of EPCs proliferation. This provides experimental evidence for further study of the combined treatment of VEGF165 gene and EPCs for ischemic disease.