Study Of Adenoassociated Virus-Mediated Cotransfer Of HTGF-β1 And HTGF-β3 Encoding Genes To The Degenerative Nucleus Pulposus Cells Of Rabbits: It's Biological Effects On Matrix Repair In Vivo

Objectives: Adeno-associated virus was applied to mediate hTGF-β1 and hTGF-β3 encoding genes to the degenerative nucleus pulposus cells of rabbits, the expression and biological effects on matrix repair of therapeutic genes were assessed.Methods: Rabbit models of disc degeneration were estabolished by injecting the N-terminal 30-kDa fibronectin fragment (Fn-f), 4 weeks later, saline with or without virus was injected directly into 112 lumbar discs of 28 skeletally mature New Zealand white rabbits. Group 1(n=8)received the rAAV2-hTGFβ1 or PBS only(self-control);Group 2 (n=6) received adeno-associated virus containing hTGFβ3 gene or saline only (self-control);Group 3 (n=6) recived rAAV2-hTGFβ1 and rAAV2-hTGFβ3, PBS serves as self-control;Group 4 (n=8) recived EGFP or saline(self-control).Two rabbits of group 1 were killed 1 week after injection, immunohistochemical staining for hTGF-β1 was performed on the nucleus pulposus (NP) tissues. NP tissues of two rabbits of group 4 were harvested for the observation of EGFP in the 12th week after gene transfer. NP tissues from the remaining rabbits were cultured and decomposed to quantify the biochemical changes of the matrix using ³⁵S-sulfate incorporation assay and western blot detection.Results: (1) Discs injected with rAAV2-hTGFβ1 exhibited extensive and intense positive immunostaining for hTGFβ1 than the self-control discs;(2) Fluorescence was observed in NP in the 12th weeks after transfer. (3)The nucleus pulposus tissues transferred with rAAV2-hTGFβ1, rAAV2-hTGFβ3 encoding gene or two above genes all exhibited increased synthesis of collagen â…¡ type and proteoglycan over that from self-control discs, the difference is statistically significant;matrix synthesis of discs received PBS and discs received rAAV2-EGFP didn't show statistic differences.(4)The expression of collagen â…¡ type and proteoglycan in discs transferred with two genes higher than that in discs with single gene transfer, the difference showed statistic significances. Conclusion: Findings showed that the discs injected with rAAV2 mediated therapeutic genes highly expressed the target proteins and kept for more than 12 weeks, which suggested that adenoassociated virus be an valid vector for the transfer of the exogenous genes to the degenerative disc. The therapeutic factor hTGF-β1 or hTGF-β3 efficiently affected the matrix repair of the degenerative disc, and they could achieve a synergistic effect on the regeneration of disc degeneration.