The present study determines whether the in vivo injection of TGFβ1 and CTGF mediated by AAV2 to transfect nucleus pulposus cells in degenerative lumbar discs can reverse the biological effects of rhesus lumbar disc degeneration. A total of 42 lumbar discs obtained from six rhesus monkeys were classified into three groups: experimental group, control group, and blank group. Degenerative lumbar discs were respectively injected with double gene-transfected human nucleus pulposus cells using minimally invasive techniques. Immumohistochemical staining, RT-PCR, and western blot were performed to observe the biological effects of double genetransfected human nucleus pulposus cells in degenerative lumbar discs on rhesus lumbar disc degeneration. At 4, 8, and 12 weeks after the transplantation of nucleus pulposus cells, the expression levels of TGF-ß1, CTGF, proteoglycan mRNA, and type-II collagen were detected by RT-PCR. The values of immumohistochemical staining and RT-PCR in the experimental group increased at 8 weeks, decreased with time at 12 weeks, and remained greater than the values in the control group, and the differences were statistically significant (P < .05). The western blot revealed that the values in the experimental group decreased with time, but remained greater than those in the PBS control group and blank control group, and the differences were statistically significant (P < .05). The double gene-transfection of human nucleus pulposus cells in degenerative lumbar discs mediated by rAAV2 can be continuously expressed in vivo after transplantation in lumbar discs of rhesus monkeys, and promotes the synthesis of proteoglycan and type II collagen, achieving the treatment purpose. K E Y W O R D S

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This study aims to detect the biological effects of matrix metalloproteinase 3 (MMP3) and SRY-related high mobility group-box gene 9 (SOX9) gene regulation in the human intervertebral disc degeneration of nucleus pulposus cells mediated by lentiviral vectors in vitro. The culture and development of human degenerated intervertebral disc nucleus pulposus cells was performed. The cell morphology and structure were observed, as identified by hematoxylin and eosin (H&E) staining. The experiment was divided into five groups: blank control group, SOX9 gene overexpression group, MMP3 gene silencing group, SOX9 gene overexpression+MMP3 gene silencing group, and blank vector group. RT-PCR and western blot were performed to detect the influence of MMP3 gene silencing and SOX9 gene overexpression on degenerated human intervertebral disc nucleus pulposus cells, include the secretion of polysaccharide and expression of type II collagen, at the mRNA and protein level. The originally generated degenerated human intervertebral disc nucleus pulposus cells had the same cellular morphology. The MTT assay revealed that the blank group and blank vector group had no statistical significance in cell proliferation. The MMP3 gene silencing and SOX9 gene overexpression could promote the proliferation of degenerated human intervertebral disc nucleus pulposus cells, when compared with the blank control group and blank vector group, and this more obvious in the MMP3 gene silencing group and SOX9 gene overexpression group. RT-PCR and western blot revealed that MMP3 gene silencing and SOX9 gene overexpression can promote cells to secrete polysaccharide and type-II collagen (P < 0.05). MMP3 gene silencing and SOX9 gene overexpression can promote the proliferation of degenerated human intervertebral disc nucleus pulposus cells, and their ability to secrete polysaccharide and type-II collagen.