Implantation Of Mscs In Nhap-polysaccharide-protein3D Active Network By Cxcr4Modification

Objective To construct recombinant adenovirus vector carrying CXCR4gene and theexpression of verification, screening and obtaining the high expression of CXCR4inMSCs, composite materials, to observe the effects of CXCR4gene transfection on theMSCs to the SDF-1chemotactic ability. To provide a certain theories basis and technicalfor the treatment of large bone defects.Methods1Contribute recombinant adenovirus vector with CXCR4, and plasmidand titer identification.2Cultured rat mesenchymal stem cells （MSCs） and screenedthe MOI. Expression of CXCR4in MSCs was verified by RT-PCR and Western-blot, celltransfection rate was detected by flow cytometry.3Three dimensional materialsactivity was detected by MTT cell compatibility.4MSCs were infected72h and culturedin Scaffold materials from transwell plate in upper, lower adding complete mediumcontaining different concentrations of SDF-1α, after co-cultured3d, observing MSCs in-depth material growth by HE staining, validated of the SDF-1/CXCR4pathway inthe scaffold cells.Results1The recombinant adenovirus vector carrying CXCR4gene was constructedsuccessfully and the plasmid and vector identification were made, titre was2×1010PFU/ml.2MSCs were isolated from the bone shaft of femurs of three week-old male mouse. Underthe inverted phase contrast microscope, most of MSCs were seeded round and spherical,then turned into spindle. About8d~10d later, the cultured cells can cover bottom flasks andmay covered with the bottle wall, swirling. The3rd passage cells were used in thesubsequent cultures.3By fluorescence microscopy and flow cytometry to detect and selectthe best adenovirus vector multiplicity of infection（MOI）of50. By RT-PCR andwestern-blot assay the expression of CXCR4cells after infection increased significantly.4Using MTT method to test cell compatibility: MSCs grew very well in materials. There isno statistical difference in the experimental group and control group（P>0.05）.5Theresult of transwell expriment: the MSCs invaded up to600μm in the group without SDF-1,the empty plasmid transfection group and the untransfected group, whereas CXCR4-overexpressing MSCs invaded up to800μm within3days. With the increasingconcentration of SDF-1α, MSCs invaded deeply. When the concentration of SDF-1α is200 μg/L, MSCs can invade as far as1200μm.Conclusion1By recombinant adenovirus vector carrying CXCR4gene in infectedcells to obtain the MSCs with high expression of CXCR4.2Experimental results showthat SDF-1can induce MSCs containing chemokine CXCR4gene, making it more deeplyinvolved in bone tissue engineering material, and participating in osteogenesis.