Adeno-associated virus-mediated BMP-7 and SOX9 in vitro co-transfection of human degenerative intervertebral disc cells.

Bone morphogenetic protein-7 (BMP-7) and SOX9 are important transcription factors in chondrogenesis. In this study, we examined the biological function of the adeno-associated virus (AAV)-mediated BMP-7 and SOX9 double gene in vitro co-transfection of nucleus pulposus cells of human degenerative intervertebral disc. Human intervertebral disc nucleus pulposus cells were cultured in vitro and subcultured for 5 generations. Using rAAV-BMP-7 and rAAV-SOX9 AAV2-type AAV viruses, the cells were divided into 4 groups: blank normal, BMP-7 transfection, SOX9 transfection, and BMP-7 and SOX9 co-transfection. After 48 h, expression of type II collagen and its mRNA in nucleus pulposus cells was determined. The expression of type II collagen in BMP-7 transfection, SOX9 transfection, and co-transfection groups was up-regulated to varying degrees compared to the blank control group. The type II collagen mRNA level expression in the co-transfection group was significantly higher than in other groups (P < 0.05). AAV-mediated BMP-7 and SOX9 in vitro co-transfection can promote the synthesis of type II collagen in nucleus pulposus cells in the human degenerative intervertebral disc. Double-gene therapy has a synergistic effect in the treatment of intervertebral disc degeneration.

Co-transfection of adeno-associated virus-mediated human vascular endothelial growth factor165 and transforming growth factor-ß1 into annulus fibrosus cells of rabbit degenerative intervertebral discs.

Intervertebral disc degeneration is a common condition that may lead to low back pain and radiculopathy. Understanding the pathophysiology and cellular and molecular events of degenerative disc disease has resulted in the proposal of a gene therapy approach to halt and reverse disc degeneration. We explored the feasibility of reversing intervertebral disc degeneration using human vascular endothelial growth factor165 (hVEGF165) and transforming growth factor-ß1 (TGF-ß1) gene therapy. hVEGF165 complementary DNA was obtained from pcDNA3(+)-hVEGF165 and cloned into adeno-associated virus (AAV)-pSNAV plasmids to construct the recombinant plasmid, AAV-pSNAV-hVEGF165. After identification through restriction enzyme digestion and DNA sequencing, the AAV-pSNAV-hVEGF165 was transfected into HEK293 cells and vascular endothelial cells. Protein expression of hVEGF165 was detected using a fluorescent immunohistochemical assay, and the effect of hVEGF165 on vascular endothelial cell proliferation was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Packaged AAV-hVEGF165 and AAV-TGF-ß1 were co-transfected into the annulus fibrosus cells of degenerative intervertebral discs. hVEGF165 and TGF-ß1 expression by annulus fibrosus cells and the effect of the co-transfection on the level of collagen type I protein expression by annulus fibrosus cells were detected with Western blot. The results of restriction enzyme digestion and DNA sequencing confirmed that AAV-pSNAV-hVEGF165 plasmids were constructed. The fluorescent immunohistochemical results confirmed hVEGF165 protein expression. The MTT results showed that the hVEGF165 protein promoted vascular endothelial cell proliferation. Biologically active AAV-hVEGF165 and AAV-TGF-ß1 were successfully constructed. Western blot confirmed hVEGF165 and TGF-ß1 expression in annulus fibrosus cells and demonstrated that the level of collagen type I protein expression was significantly higher in annulus fibrosus cells co-transfected with both AAV-hVEGF165 and AAV-TGF-ß1 compared with that in cells transfected with AAV-hVEGF165 or AAV-TGF-ß1 alone. hVEGF165 has a synergistic effect with TGF-ß1 that promotes the expression of collagen type I protein in annulus fibrosus cells from degenerative intervertebral discs.