| Purpose: To investigate whether genetic modification effects on the biological character of dental pulp stem cells(DPSCs), we used adenovirus vectors transfected the gene Scleraxis(SCX) into DPSCs.Methods: 1.We isolated DPSCs from dental pulp tissue by enzymatic digestion method in vitro.In addition, the osteogenic potential of DPSCs were investigated by Alizarin red staining after osteo-induction for 4 weeks and the chondrogenic potential of DPSCs were investigated by alcian blue staining after chondro-induction for 4 weeks. 2. To identify sources of cells, DPSCs were detected the Vimentin by immunocytochemistry.3.TO verify the character of stemness, DPSCs which was isolated and cultured by the method introduced above, was detected the surface markers CD146 and Stro-l by immunofluorescence.4. Adenovirus vector carried SCX gene was structured in vitro and transfected into DPSCs.we made a preliminary experiment to confirm the optimum multiplicity of infection(MOI).5. Effective expression of SCX was observed by real time PCR and Western Blot, which proved the successful transfection of SCX into DPSCs. 6. We compared the difference in the proliferation capability between the blank and transfected DPSCs by MTT.7.To investigate the effect of genetic modification effects on the multipotention of DPSCs towards osteoblast, odontoblast and cementoblast, real-time PCR test was applied to detect the expression of TNC, TNMD, Osx, Runx2, CAP, CP-23, and DMP-1 between the blank and transfected DPSCs at 3rd, and 7th days after transfection.Results:1.DPSCs that isolated by enzyme digestion method endowed with a superior proliferative capacity and clonogenicity.2.Immunofluorescence test indicated that the cell isolated from dental pulp express CD146 and Strol-1,and theimmunocytochemistry indicated that the cultured cells showed the express of Vimentin.3. After osteogenesis induced for 4 weeks, DPSCs were treated with alizarin red staining and mineralization nodule formation was observed.Meanwhile, DPSCs was treated with chondro-induced medium for 4weeks using “cell mass cultivation” and the deposition of cartilage matrix was observed by Alcian blue staining. 4.Afer DPSCs transfected for 24 hour, green fluorescene protein could be observed under fluorescence microscope.with the level of MOI increasing, more DPSCs could be seem the green linght under the fluorescence microscope. Menntime,,more than 80 percent transfected cells when the value of MOI setted from 100 to 200.At the 2nd day almost all DPSCs could see the green fluorescene.5.the western blot and real time PCR indicated the succefully transfection of SCX into DPSCs.And the gene SCX was consistently over-expression in DPSCs.6.No obvious alteration in cell morphology was observed under optical and fluorescent microscope.No significant difference in the proliferation was observed by MTT test. 7. Real-time PCR indicated that the expression of TNC, TNMD, Osx, CP-23, DMP1 enhanced at day3,however, the express of Runx2, CAP down regulated at 3rd day. There was significant difference between them in the expression of Osx(P<0.05). Data of Realtime PCR showed that the levels of TNMD,CAP,CP-23,DMP1 in transfected DPSCs were higher than those in blank(P<0.05) at 7th day, meanwhile, there was significant difference between them in the expression of CAP,DMP1(P<0.05).Conclusion: 1,The cell isolated from dental pulp tissue by enzymatic digestion method in vitro showed the character of stemness,and DPSCs have a multipotention to induce into osteblast and chondroblast.2,The adenvirus successfully transfected the gene SCX into DPSCs,meanwhile, the gene SCX was consistently over-expression in DPSCs.The treatment did not significantly affected the proliferation of DPSCs.3.The cultured cells showed characteristics of mesoderm by immunocytochemistry.Transfected DPSCs express a functionally SCX protein,which may enhanced the differentiation towards tendon.4,The DPSCs may be a promising cell in the regeneration of periodontal tissue... |
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