title: Co-transfection of adeno-associated virus-mediated human vascular endothelial growth factor165 and transforming growth factor-β1 into annulus fibrosus cells of rabbit degenerative intervertebral discs.
abstract: 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-β1 (TGF-β1) 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-β1 were co-transfected into the annulus fibrosus cells of degenerative intervertebral discs. hVEGF165 and TGF-β1 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-β1 were successfully constructed. Western blot confirmed hVEGF165 and TGF-β1 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-β1 compared with that in cells transfected with AAV-hVEGF165 or AAV-TGF-β1 alone. hVEGF165 has a synergistic effect with TGF-β1 that promotes the expression of collagen type I protein in annulus fibrosus cells from degenerative intervertebral discs.
authors.affiliation: Department of Orthopaedic Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shandong, China.
authors.forename: Y M
authors.initials: YM
authors.lastname: Xi
authors.forename: Y F
authors.initials: YF
authors.lastname: Dong
authors.forename: Z J
authors.initials: ZJ
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: Z Z
authors.initials: ZZ
authors.lastname: Diao
authors.forename: Y G
authors.initials: YG
authors.lastname: Hu
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D024042:Collagen Type I / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000235:genetics* / Q000628:therapy*; D011817:Rabbits; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A