An In Vitro Study Of Degenerated Human Lumbar Intervertebral Discs: The Biological Effects Of CTGF And TIMP1 On Nucleus Pulposus Cells Mediated By Adeno Associated Virus

Objective: To construct the recombinant plasmid of adeno-associated viruscontaining human connective tissue growth factor and tissue inhibitor ofmetalloproteinases, pSNAV₂-CTGF-IRES-TIMP1, and to package into virusparticles after detecting the biological activity in vitro, and then to evaluate thereversion possibility of CTGF and TIMP1 to human lumbar intervertebral discdegeneration by gene method.Methods: The recombinant plasmid pSNAV₂-CTGF-IRES-TIMP1 wasidentified by PCR, restriction enzymes analysis and sequencing analysis, and thentransduced into HEK293 cells by lipofectamine mediated gene transfer method. Thedouble staining immunofluorescence, Western blot, MTT and ELISA were used todetect the biological activities of CTGF and TIMP1 proteins, and then therecombinant plasmid was packaged to virus particles, rAAV₂-CTGF-IRES-TIMP1was transduced into human nucleus pulposus cells of degenerated lumbarintervertebral discs, which have been cultured and identified in vitro, and then todetect the changes of proteoglycans and collagen typeâ…¡in nucleus pulposus cells bycell morphology, miamine method, immunohistochemistry, ³⁵S incorporation assayand Antonopoulos method.Results: The recombinant plasmid was constructed successfully withcompleted correctly CTGF and TIMP1 genes, rAAV₂-CTGF-IRES-TIMP1 couldtransfect into human nucleus pulposus cells of degenerated intervertebral discs.CTGF and TIMP1 could increase the synthesis of proteoglycans and collagen typeâ…¡in nucleus pulposus cells.Conclusions: The CTGF and TIMP1 genes mediated by adeno associated viruscould delay or even reverse the degeneration of human lumbar intervertebral discsby promoting the synthesis of proteoglycans and collagen typeâ…¡.