The Migration Efficiency Of Upregulation Of SDF-1A/CXCR4 Axis On Bone Mesenchymal Stem Cells

Objective Isolation and culture bone marrow mesenchymal stem cells(BMSCs)derived from Sprague-Dawley rats to ensure a sufficient and stable source of cells.Construction of lentiviral vectors and infection of BMSCs in vitro.To obtain bone marrow mesenchymal cells overexpress CXCR4.In vitro,to investigate the migration efficiency of upregulation of sdf-1/cxcr4 axis on bone mesenchymal stem cells.Methods To isolate,culture BMSCs applying tissue adherent culture method.BMSCs were divided into three groups,they are CXCR4-GFP-BMSCs(overexpression group),GFP-BMSCs(empty virus group)and BMSCs group(control group).To construct a lentiviral vector with fluorescent protein gene and CXCR4 gene,identified by PCR and sequencing,pack viral vector and detect virus titer.To infect BMSCs with the lentiviral vector,and observed by fluorescence microscope to assure the optimal of MOI for those two groups(CXCR4-GFP-BMSCs and GFP-BMSCs),respectively.To assure the expression of CXCR4 in these three groups by PCR and Western blot.The effects of different concentrations of SDF-1(0,50,100,200 ng/ml)on BMSCs were observed by Transwell assay.The migration efficiency of BMSCs was observed.The best SDF-1 concentration was used to chemotaxize different CXCR4-expressing stem cells to detect the migration efficiency of different CXCR4-expressing stem cells.Result The lentiviral vector was successfully constructed in vitro.The CXCR4 gene was verified by PCR and gene sequencing.The optimal MOI for CXCR4-GFP-BMSCs was 30 and infection efficiency were 80%.The expression of CXCR4 in overexpressing group was higher than other two groups,vertified by Western Blot and RT-PCR.The migration efficiency of BMSCs enchanced with the increase of SDF-1 concentration,and the migration efficiencies of 100ng/ml and 200ng/ml were same.Chemotactic by same SDF-1 concentration,the migration efficiency of BMSCs overexpressing CXCR4 was significantly higher than that of the other groups.Conclusion ① High purity BMSCs were obtained by whole bone marrow adherent.② Lentiviral infection is a valid and feasible method for overexpressing CXCR4 in BMSCs;③Upregulate SDF-1/CXCR4 axis can promote the efficacy of migration of BMSCs in vitro.