[Biological effects of recombinant adeno-associated virus 2 mediated human transforming growth factor beta1, encoding gene transfer to rabbit degenerative nucleus pulposus cells on proteoglycan level].
OBJECTIVE
To verify the potential of the recombinant adeno-associated virus 2 (rAAV2) vector as a strategy for human transforming growth factor beta1 (hTGF-beta1) gene transfer in degenerative intervertebral discs of rabbit, to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring.


METHODS
Rabbit models of disc degeneration were established by injecting the 25 microL fibronectin fragment (Fn-f, 1 mmol/L), 4 weeks later, saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits (male or female and weighing 1.7-2.2 kg) which were divided into 3 groups (n = 8). Group A received the 25 microL rAAV2-hTGF-beta1 (1 x 10(12) vg/mL); group B received rAAV2-enhanced green fluorescent protein (rAAV2-EGFP); and group C received PBS. Two rabbits of groups A, C were killed 1 week after injection, the immunohistochemical staining for hTGF-beta1 was performed on the slices of nucleus pulposus (NP) tissues. At 4, 8, and 12 weeks after gene transferring, NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using 35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection.


RESULTS
Immunohistochemical staining showed that extensive and intense positive immunohistochemical staining for hTGF-beta1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan, which was significantly more than that from groups B and C (P < 0.05), and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks.


CONCLUSIONS
The discs injected with rAAV2-hTGF-beta1 can highly expressed the therapeutic proteins for more than 12 weeks, it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-beta1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.
Liu
Haifei
H
Department of Orthopaedics, Traumatic and Orthopaedic Research Institute of Shandong Province, Affiliated Hospital of Medical College, Qingdao University, 266003 Qingdao Shandong, PR China.
Ning
Bin
B
Wang
Dechun
D
Hu
Yougu
Y
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D015316:Genetic Therapy; D005822:Genetic Vectors; D007403:Intervertebral Disc / Q000166:cytology*; D008297:Male; D011509:Proteoglycans / Q000096:biosynthesis*; D011817:Rabbits; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics*
D011509:Proteoglycans; D053773:Transforming Growth Factor beta1