Lentiviral transfection of pigment epithelial derived factor gene into human umbilical cord mesenchymal stem cells / 中华眼底病杂志

ABSTRACT

Objective To build the lentiviral vectors of pigment epithelial derived factor (PEDF) gene,and investigate their expression in human umbilical cord mesenchymal stem cells (hUCMSCs).Methods The PEDF lentiviral vectors (LV-PEDF) were built by DNA recombination and confirmed by DNA sequencing.hUCMSCs were transfected by LV-PEDF with MOI 10,30,50,respectively.The transfection efficiency was observed under fluorescence microscope.Cell immunofluorescence,immunocytochemistry and real-time PCR methods were used for detecting the expression of PEDF and VEGF.Results The PEDF cDNA was sub-cloned into pCDH-CMV-MCS-EF 1-copGFP vector successfully.DNA sequencing analysis confirmed that PEDF gene sequence was exactly the same with that reported in GenBank.pCDH-PEDF infected cells could show green fluorescence under fluorescence microscope.The transfection efficiency was 72.1％ in PEDF-MSCs.Immunofluorescence and immunochemical staining confirmed that PEDF protein was overexpressed in hUCMSCs.The relative expression of PEDF mRNA in experimental group and control group was (0.170±0.028) and (0.015 ± 0.007) respectively by RT-PCR,the difference was statistically significant (P＜0.00 1).The relative expression levels of VEGF mRNA in the two groups were (0.265 ± 0.022) and (0.285 ± 0.049),respectively,with no significant difference (P＞0.05).Conclusions We successfully built a lentivius vector carrying PEDF gene and obtained hUCMSCs with overexpressed PEDF.