A Study Of Degenerated Rhesus Monkey Lumbar Intervertebral Discs: The Biological Effects Of CTGF And TIMP₁ Mediated By Adeno Associated Virus In Vitro And Vivo

Objective:To study the biological effect of connective tissue growth factor and tissue inhibitor of metalloproteinases 1 mediated by adeno-associated virus on rhesus monkey lumbar intervertebral discs cell in vitro and vivo.Methods:In vitro:The nucleus pulposus cells of rhesus monkey lumbar intervertebral disc were cultured through enzyme digestion.The rAAV₂-EGFP was used to infect the cells to make sure the proper MOI of the AAV.The nucleus pulposus cells were infected by rAAV₂-CTGF,rAAV₂-TIMP₁ and rAAV₂-CTGF-IRES-TIMP₁ using the proper MOI of the AAV.The biological effect was detected through RT-PCR,³⁵S incorporation assay and Safranine-O.In vivo:To establish a novel model of lumbar disc degeneration in the rhesus monkey using percutaneous needle puncture guided by CT. The rAAV₂-CTGF-IRES-TIMP₁ was injected percutaneous with minimally invasive technique.The expression of CTGF and TIMP₁ was determined through RT-PCR,Western blot.The biological effect of rAAV₂-CTGF-IRES-TIMP₁ was examined by RT-PCR,³⁵S incorporation assay and tissue stainResults:In vitro:The proper MOI is 10⁶ through rAAV₂-EGFP infecting nucleus pulposus cells of rhesus monkey lumbar intervertebral disc.rAAV₂-CTGF,rAAV₂-TIMP₁ and rAAV₂-CTGF-IRES-TIMP₁ could transfect into nucleus pulposus cells and CTGF, TIMP₁ also could express.CTGF could increase the synthesis of proteoglycans and collagen typeâ…¡in nucleus pulposus cells.TIMP₁ also could increase the synthesis of proteoglycans.The best effect for the increasing the matrix synthesis is rAAV₂-CTGF-IRES-TIMP₁.In vivo:A model of lumbar disc degeneration on the early stage in the rhesus monkey with minimally invasive technique was establishend successfully.The CTGF and TIMP1 could express 24 weeks in vivo after transferring into the intervertebral disc,which also could increase the matrix synthesis.Conclusions:The CTGF and TIMP₁ genes mediated by adeno-associated virus could increase the matrix synthesis.The best effect for the creasing matrix synthesis is rAAV₂-CTGF-IRES-TIMP₁.