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

Study of green fluorescent protein gene transfection in endothelial progenitor cells mediated by lentivirus vector

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（P0.05）.Compared with GFP untransfected group,the cell growth curve of MOI 1∶ 50 group had no significant difference（P0.05）.It showed significant arrest and damage of cell growth in MOI 1∶ 100 GFP transfection group.Conclusions GFP can be effectively transferred into EPCs under HIV-1 lentivirus vector mediation.MOI 1∶ 50 has better transfection efficiency and no influence to cell growth.