慢病毒载体介导绿色荧光蛋白基因转染血管内皮祖细胞的实验研究

摘要

目的 探讨HIV-1来源的慢病毒载体介导绿色荧光蛋白(GFP)基因转染血管内皮祖细胞(EPCs)的可行性和方法.方法 用梯度密度离心法分离人脐带血内皮祖细胞,在EGM-2培养基中培养.用细胞免疫荧光染色和流式细胞仪检测其表达情况.以HIV-1来源的慢病毒为载体、以GFP基因为目的 基因转染EPCs,MTT法检测不同病毒滴度(MOI)时细胞增殖情况并观察转染率.结果 单个核细胞经EGM-2培养基培养1周后即分化成EPCs.GFP转染后48 h细胞即发出绿色荧光.MOI 1:10转染组细胞转染率低于MOI 1:50组(P＜0.05).MOI 1:50转染后的细胞与未转染GFP组比较,生长曲线无明显差异(P＞0.05).MOI 1:100组转染后细胞的增殖处于停滞状态.结论 采用HIV-1来源的慢病毒载体介导GFP基因转染标记EPCs是可行的.以MOI 1:50进行转染对细胞生长影响小,转染效率高.%Objective To understand the feasibility and methods of green fluorescent protein (GFP) transfection in endothelial progenitor cells (EPCs) mediated by lentivirus vector HIV-1.Methods EPCs harvested from umbilical cord blood were isolated by gradient density centrifugation and cultured in EGM-2 medium.The cells were identified by immunofluorescence staining and flow cytometry.Cell proliferation and transfection efficiency were assayed by MTT method.Results The mononuclear cells cultured in EGM-2 medium were confirmed to be EPCs after 1 week.After transfection of 48 hours, the cells demonstrated green fluorescence.It had no significant difference in cell proliferation between multiplicity of infection (MOI) 1:10 group and MOI 1:50 group.GFP-positive rate in MOI 1:10 group was significant lower than that in MOI 1:50 group (P ＜ 0.05).Compared with GFP untransfected group, the cell growth curve of MOl 1:50 group had no significant difference (P ＞ 0.05).It showed significant arrest and damage of cell growth in MOl 1: 100 GFP transfection group.Conclusions GFP can be effectively transferred into EPCs under HIV-1 lentivirus vector mediation.MOl 1: 50 has better transfection efficiency and no influence to cell growth.