The Construction Of AAV-hVEGF₁₆₅ And The Biological Effect Of HVEGF₁₆₅ And TGFβ1 In Annulus Fibrosus Cell Of Intervertebral Disc

Objective: To construct the viral vector AAV-hVEGF₁₆₅, and to detect its expression in annulus fibrosus cell of intervertebral disc, then to observe the cotransfection bioactivity of AAV-hVEGF₁₆₅ and AAV-TGFβ1 to annulus fibrosus cell. Further to evaluate the hVEGF₁₆₅ reversion possibility of intervertebral disc degeneration by gene method.Methods: The hVEGF₁₆₅cDNA obtained from plasmid pcDNA3(+)-hVEGF₁₆₅ was subcloned into the packaging plasmid pSNAV of AAV by molecular clone ways. The recombinant plasmid pSNAV-hVEGF₁₆₅ was identified by restriction enzymes analysis and sequencing analysis, and then transfered to the HEK293 cell and VEC by lipofectamine mediated gene transfer method. The protein hVEGF₁₆₅ was detected by immunofluorescence for immunocytochemistry and was explored the influence to VEC's proliferation by MTT way. Whereafter the AAV-hVEGF₁₆₅ was packaged by Benyuan Zhengyang Corporation.Further AAV-hVEGF₁₆₅ and AAV-TGFpi were cotransfected into annulus fibrosus cell of intervertebral disc, then the expression of hVEGF₁₆₅ and TGFβ1 were detected by Western blotting, and the expression change of collagen I in annulus fibrosus cell was also explored by the same method.Results: The recombinant pSNAV-hVEGF₁₆₅ was completely constructed and confirmed by restriction enzymes analysis and sequencing analysis. The protein hVEGF₁₆₅ was detected by immunofluorescence for immunocytochemistry in experimental group, but in control group the protein hVEGF₁₆₅ was not expressed, and hVEGF₁₆₅ could promote VEC's proliferation. The bioactive AAV-hVEGF₁₆₅ was packaged by Benyuan Zhengyang Corporation, and the AAV-hVEGF₁₆₅ was successfully constructed. The expression of AAV-hVEGF₁₆₅ and AAV-TGFβ1 were manifested in annulus fibrosus cell by Western blotting, and the expression of collagen I in annulus fibrosus cell cotransfected by AAV-hVEGF₁₆₅ and AAV-TGFβ1 was markedly more than that of the control cell or monogenic transfected cell.Conclusion: The constructed AAV-hVEGF₁₆₅ could express in vitro, and AAV-hVEGF₁₆₅ could cooperate with AAV-TGFβ1 to promote the expression of collagen I .hVEGF₁₆₅ could possibly reverse the early degeneration of intervetebral disc.