Study of recombinant adeno-associated virus 2-mediated cotransfer of hTGF-β1 and hTGF-β3 encoding genes to the rabbit degenerative nucleus pulposus cells / 中华骨科杂志

ABSTRACT

Objective 1) To verify the the potential of the adenoassociated viral vector as a strategy for intradiscal gene transfer in degenerative rabbit intervertebral discs. 2) To investigate the gene transduction efficacy and to quantify the biologic effects on the matrix synthesis after single gene transfer and combined gene transfer. Methods Rabbit models of disc degeneration were established by injecting the N-terminal 30×103 fibronectin fragment (Fn-f), 4 weeks later, saline with or without virus was injected directly into 144 lumbar discs of 36 skeletally mature New Zealand white rabbits. Group A (n =8) received the rAAV2-hTGFβ1; Group B (n=6) received rAAV2-hTGFβ3;Group C (n=6) recived rAAV2-hTGFβ1 and rAAV2-hTGFβ3; Group D (n=8) recived rAAV2-EGFP as the experimental control. Group E (n=8) recived PBS as the blank control. Two rabbits of the group A and group E were sacrefied 1 week after injection, immunohistochemical staining for hTGF-β1 was performed on the slices of nucleus pulposus (NP) tissues. On 4,8 and 12 weeks after gene transferring, NP tissues were cultured or decomposed to quantify the biochemical changes of the matrix using 35S-sulfate incorporation assay and western blot detection. The expression of EGFP was observed 12 weeks after injection. Results Discs in group A exhibited extensive and intense positive immunostaining for hTGF-β1 than the control discs in group E 1 week after gene transferring. The nucleus pulposus tissues in group A, B and C exhibited a 1.28-2.06 fold increase in proteoglycan synthesis and a 1.25-1.73 fold increase in collagen type Ⅱ production over those in group E (P＜0.05 or P＜0.01).Combination of two gene transfer in group C makes a significantly increased level of proteoglycan (1.195-1.290 fold)and collagen type Ⅱ (1.152-1.219 fold) than single gene transfer in group A and B(P＜0.05 or P＜0.01).No statistic differences shows between A group and B group. The difference of the matrix synthesis between group D and group E was also not statistically significant (P＞0.05). Extensive and intensive green fluorescence was observed on the slice of nucleus pulposus tissues received rAAV2-EGFP 12 weeks after gene delivery. The expression of EGFP kept for more than 12 weeks. Conclusion Findings showed that the disc tissue injected with rAAV2 mediated genes highly expressed the therapeutic proteins from 1 week to more than 12 weeks after delivery. It is suggested that adenoassociated virus be an valid vector for the transfer of the exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 and hTGF-β3 could efficiently increase the synthesis of proteoglycan and collagen type Ⅱ in the degenerative NP cells and combined transfer of two genes was more effective than single gene transfer. The two factors have an positive synergistic effects.