Study On Rat Bone Marrow Stromal Cells Cultured In Vitro And Transfected With CEMP1 Gene

Objective: To construct a recombinant lentiviral vector containing human CEMP1 gene(LV-CEMP1-EGFP)and detect its effect on rat bone marrow stromal cells(rBMSCs).Methods: 1.rBMSCs were isolated and cultured in vitro and then surface markers of rBMSCs were detected by flow cytometry.Osteogenic and adipogenic differentiation were evaluated after rBMSCs were cultured in osteogenic medium and dipogenic medium.2.Recombinant lentiviral vector containing human CEMP1 was constructed and confirmed by RT-PCR and gene sequence analysis.3.Transfection efficiency was detected by fluorescence detection and flow cytometry 72 hours after transfection.The expression of CEMP1 in rBMSCs was detected by RT-PCR,Real-time PCR,Western Blot and immunocytochemistry.4.The expression of cementoblast-associated genes in transfected rBMSCs were detected to evaluate the effect of CEMP1 gene on rBMSCs.Results:1.rBMSCs were successfully isolated and cultured in vitro,flow cytometry showed high expression of CD29 and CD90 on the surface of rBMSCs.2.rBMSCs were osteogenic induced for 21 days and alizarin red staining showed mineralized nodules.Also rBMSCs were adipogenic induced for 21 days and oil red O staining showed lipid droplets.3.Successful construction of LV-CEMP1-EGFP was confirmed by RT-PCR and gene sequence analysis.4.The rBMSCs transfected with LV-CEMP1-EGFP showed a lot of green fluorescence under fluorescent microscope and high transfection efficiency 72 hours after transfection.5.RT-PCR,Real-time PCR,Western Blot and immunocytochemistry showed high expression of CEMP1 in rBMSCs transfected with LV-CEMP1-EGFP.6.LV-CEMP1-EGFP group showed higher expression of BSP and OPN than LV-EGFP group and control group,the difference is of statistical significance(P<0.05).Conclusion: The lentiviral vector carrying human CEMP1 gene was successfully constructed and transfected into rBMSCs.CEMP1 can promote the expression of cementoblast-related genes in rBMSCs.