Differentiation Of Mesenchymal Stem Cells Into Nucleus Pulposus-like Cells By Transfected With Sox9 Eukaryotic Expression Vector In Vitro

OBJECTIVE To investigate the effects of the recombinant plasmid pcDNA3.1IE-SOX9Flag on differentiation of rabbit mesenchymal stem cells into Intervertebral disc cells.METHODS The eukaryotic expression vector of SOX9 was constructed. Rabbit MSCs(mesenchymal stem cells )were isolated and cultured from one month old New Zealand white rabbits and induced into osteogenetic cells in the osteogenesis supplement medium and detected cell surface markers by flow cytometer. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were transfected with recombinant plasmid pcDNA3.1IE-SOX9Flag and the control group in which the cells were treated with the media without recombinant plasmid .After selected by G418 in two weeks, the cells were harvested and RT-PCR was employed to assay SOX9 and typeⅡcollagen mRNA expression in MSCs. The expression of SOX9 was assayed by Western-blot and typeⅡcollagen was also determined by immunohistochemical staining. RESULTS The SOX9 eukaryotic expression vector was constructed successfully. The cells that were induced into osteogenetic cells were positive for the ALP staining. What's more, CD44 expression was positive but CD34 and CD45 was negative. The transfection efficiency is 34.32%±1.75% which was obtained at 72h after transfected. After two weeks, the modality of cells which were tansfected with SOX9 turned to polygonal and elliptical. And the cell proliferation was gradually slow. RT-PCR identification showed that SOX9 gene and typeⅡcollagen mRNA expression was positive in transfected group, the control groups were negative. Western-blot detected SOX9 protein expression in transfected group but no expression in the control groups. At 2 weeks after transfection, the cells in transfected group were positive for immunohistochemical staining of typeⅡcollagen.CONCLUSION The recombinant plasmid pcDNA3.1IE-SOX9Flag can be successfully transfected into rMSCs that inducing MSCs differentiated into Nucleus Pulposus-like cells, which provided the foundation of using cells transplantation therapy for Intervertebral disk degeneration.