Over-expression Of SDF-1α Promote The Migration Of Bone Marrow Mesenchymal Stem Cells In Vitro

ObjectivePrevious Researches showed that SDF-1/ CXCR4 axis can not only promote the migration of the transplanted BMSCs to the damaged tissue, but also inhibit the apoptosis of BMSCs,,increase the survival rate.and promote the proliferation of them,which could improve the homing efficiency of BMSCs to the damaged tissue in many ways. Therefore, we may envisaged that the exogenous SDF-1 gene transfer into BMSCs would increase the expression of SDF-1 in BMSCs thus enhancing the effect of migration in BMSCs.Methods1. SDF-1α gene was cloned into the transfer vector of lentivirus. Then the vectors was treated with PCR, restriction enzyme digestion and sequencing;2.p NL-SDF-1α-IRES2-EGFP, p NL-IRES2-EGFP and GV-118-SDF-1α-si RNA along with corresponding vectors were cotransfected into 293 T cells to package lentivirus particles. Then the particles were concentrated to determine viral titer and function. Lentiviral transduction was carried out to transfer SDF-1α-lentivirus、si RNA-lentivirus and null-lentivirus into BMSCs, respectively. The BMSCs were selected for stable integrants by using EGFP reporter gene. The expression of SDF-1α gene in BMSCs was evaluated by RT-PCR and Western Blotting;3.The influence of SDF-1α on migration of BMSCs was evaluated by Transwell migration experiment. After being incubated with anti SDF-1α monoclonal antibody, the ability of migration of BMSCs were detected as well.Results1,The result of restriction enzyme digestion and sequencing showed that the full-length fragment of SDF-1α gene was successfully cloned into the transfer vector of lentivirus;2, After 48 h of Lentivirus transfection,a large number of EGFP are expressed by 293 T cells under inverted fluorescence microscope especially in SDF-1α-BMSCs group, si RNA-BMSCs group and null-BMSCs group.Results show that the expression of SDF-1α was significantly higher in SDF-1α-BMSCs group as compared with null-BMSCs group by RT-PCR and Western Blotting,while that of si RNA-BMSCs group is the lowest;3,Transwell migration experiment suggests that SDF-1α can obviously promote the transmembrane migration of BMSCs.The migration ability of the BMSCs incubated with anti-SDF-1α monoclonal antibody was restrain markedly.ConclusionsUsing the successfully constructed lentivirus vector of overexpression of SDF-1α and SDF-1α gene silencing to, infect BMSCs,. The expression of SDF-1α m RNA and protein were detected,by RT-PCR and Western Blot.Conclusions come that the overexpression of SDF-1α can enhance BMSCs in SDF-1α gene expression, and SDF-1α gene silence can inhibit BMSCs SDF-1α gene expression, Transwell migration experiments showe that overexpression of SDF-1α promote BMSCs migration across the membrane, while SDF-1α gene silence cause significantly inhibited of migration.After adding SDF-1α antibodies, transmembrane migration of BMSCs was significantly reduced.