The Study Of Rat Bone Mesenchymal Stem Cells Transfected By OPG With Lentiviral Vector In Viro

Objective:To investigate the osteoprotegerin (OPG)gene expression and analyzed the biological activity of BMSCs after transfected with PGC-FU-OPG-RFP in vitro.Methods:The BMSCs were isolated from bone marrow in SD rats about four-week-old. When training to third-generation transfer, BMSCs were divided into three groups: non-transfection group, blank Lentiviral vector group and with OPG lentivirus vector group. the cells in the non-transfection group received no special treatment, while cells in blank Lentiviral vector group were transfected with PGC-FU-RFP and cells in with OPG lentivirus vector group were transfected with PGC-FU-OPG-RFP. At48hours after transfected, fluorescent expression were detected under immuno-fluorescence microscopy; Lentiviral transfection efficiency were detected by flow cytometry; the proliferativity cells were assayed by methabenzthiazuron(MTT) method; OPG protein in transfected cells were determined by Western Blot.Results:1. Successfully developed a third generation of SD rat BMSCs,adherent BMSCs in the distribution in the bottom of the bottle, long fusiform, polygonal, grow well.2. The third generation BMSCs were transfected By lentivirus vector with MOI:10,50,100. At48hours after transfected, fluorescent expression were detected and non-transfection group did not see. Trans-fection efficiency respectively is12.0%,41.8%,70.0%。3. The transfected cell in good condition, proliferation without obvious changes and the three groups no significant difference was evaluated by MTT.4. Western Blot:72kDa feature Strip were detected in the group of transfected with PGC-FU-OPG-RFP. It is equal to the size of OPG-RFP fusion protein (-44kDa+28kDa=72kDa), We can determine the expression of osteoprotegerin.Conclusion:1. BMSCs were transfected with lentiviralvector successfully.When MOI is100,transfer rate of up to70%.2. The transfected cell is not significant difference with non-transfection ones in proliferation. OPG protein in transfected cells were determined by Western Blot.