The Study Of Green Fluorescent Protein Gene Transfection In Endothelial Progenitor Cells Mediated By Lentivirus Vector

Objective:To study the isolation, culture, amplification and identification of endothelial progenitor cell(EPCs).To understand the feasibility and methods of green fluorescent protein(GFP)transfection in endothelial progenitor cells by mediated by HIV-1 lentivirus vector.Methods:Endothelial progenitor cells harvested from umbilical cord blood were isolated by gradient density centrifugation and cultured in EGM-2 medium.The cells were identified by CD133,CD34,VEGFR-2 immunofluorescence staining.Different concentrations of lentiviral vector (multiplicity of infection MOI:1:10,1:50, 1:100) were used to transfect green fluorescent protein (GFP) into EPCs.The efficiency of cell transfection was assayed by different viral titers in MTT. The cell proliferation was observed in 10 days transfected.statistic analysis was tested by t test or test analysis of variance and Chi-square methods.P<0.05 was thought to be significant difference.Results:The mononuclear cells cultured in EGM-2 were confirmed to be EPCs by positive CD133,KDR and CD34 staining. It had no significant difference in cell proliferation between MOI 1:10 group and MOI 1:50 group.After 48 hours transfected,GFP-positive rate was(35.2±2.75)% in MOI 1:10 group, which was significant lower than that in MOI 1:50 group(87.4±1.89)%(P<0.05).Compared with untransfected group, the cell growth curve of MOI 1:50 group had no significant difference (P>0.05).It showed significant arrest and damage of cell growth in MOI 1:100 transfection group. Conclusions:High purity and quantity of EPCs can be obtained by density gradient centrifugation and culture. Green fluorescent protein can be effectively transferred into endothelial progenitor cells under HIV-1 lentivirus vector mediate. MOI=1:50 transfection ratio has better efficiency.