The Biological Effect Of Bmp-7 And Sox9 In Degenerative Annulus Fibrosus Cell Of Intervertebral Disc

Objective:To construct the adeno-associated virus (pAAV-hBMP7-IRES-ZsGreen) vector with human bone morphogenetic protein 7 (BMP7) and adeno-associated virus (pAAV-hSOX9-IRES-tdTomato) vector with SOX9, and package into virus particles,and assess its biological activity and function by transducting into human disc cells in vitro.Methods:The BMP-7cDNA sequence amplified from plasmid pUC57-hBMP-7 was cloned into pack plasmid pAAV-IRES-ZsGreen. Plasmid pAAV-hBMP7-IRES-ZsGreen was constructed by pAAV-IRES-ZsGreen. The recombinant plasmid pAAV-hBMP7-IRES-ZsGreen was identified by PCR, restriction enzymesanalysis and sequencing analysis,and then package into virus particles.The package of SOX9 adeno-associated virus (pAAV-hSOX9-IRES-tdTomato) was obtained in the same way. The recombinant expression plasmid AAV-BMP-7 and AAV-SOX9 was co-transfected into human annulus fibrosus cells of degenerated lumbar intervertebral discs. Westernblot was used to detect the expression changes of collagentypeⅡ.Results:The recombinant pAAV-hBMP7-IRES-ZsGreen and pAAV-hSOX9-IRES-tdTomato were completely constructed and confirmed by restriction enzymes analysis and sequencing analysis. The protein hBMP-7 and hSOX9 were detected succesfully by immuofluorescence. AAV-BMP-7 and AAV-SOX9 were constructed successfully.The expression of AAV-hBMP-7 and AAV-hSOX9 were manifested in degenerative annulus fibrosus cell by Western blotting. Westernblot showed that the expression levels of collagen II in annulus fibrosus cell was higher after cotransfection.Conclusion:The recombinant AAV-hBMP-7 and AAV-hSOX9 could express in vitro. BMP-7 could cooperate with SOX9 to promote the expression of collagen II in vitro. which may provide the basis for in vivo experiments and gene reversion of intervertebral disc degeneration.