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Effect of rAAV2-hTGFβ1 Gene Transfer on Matrix Synthesis in an In Vivo Rabbit Disk Degeneration Model
Hai-Fei Liu1, Bin Ning, Han Zhang
1*The Department of Orthopedic Surgery, The Affiliated Hospital of Medical College, Qingdao University†The Traumatic and Orthopedic Research Institute, Shandong Province‡The Department of Orthopedic Surgery, Jinan Central Hospital, Shandong University§The Department of Internal Medicine, The Affiliated Hospital of Medical College, Qingdao University, Shandong, China.
Study Design:
In vivo gene transfer for disk regeneration.
Objective:
To evaluate the efficiency and effect of human transforming growth factor β1 (hTGFβ1) gene transfer mediated by adeno-associated virus (AAV) in a rabbit disk degeneration model induced by fibronectin fragment (Fn-f).
Summary Of Background Data:
Gene therapy for disk degeneration has been reported to be effective. Nevertheless, few investigations have targeted the degenerative nucleus pulposus (NP) cells in vivo. Fn-f-induced degeneration has been previously verified to be a useful model for the study of disk degeneration at the molecular level. AAV vector is well suited for gene transfer in the disk for its lower immunogenicity and higher safety.
Materials And Methods:
The early dedifferentiated NP cells were transfected with rAAV2-mediated enhanced green fluorescent protein (EGFP) gene in vitro. Fluorescence expression was observed 48 hours later. The rabbit disk degeneration model was established with a microinjection of Fn-f. Ninety-six degenerative disks of 24 rabbits were injected with rAAV2-hTGFβ1 (group A), rAAV2-EGFP (group B), or PBS (group C). Immunohistochemical staining for hTGFβ1 and fluorescence observation were performed at the 1- and 12-week time points, respectively. 35S-sulfate incorporation assay and Western blot analysis were used to measure the synthesis of proteoglycan and collagen type II at 4-, 8-, and 12-week time points.
Results:
The dedifferentiated NP cells exhibited intensive fluorescence expression in vitro, with a transfection rate of 90%. In vivo, disks in group A showed enhanced positive hTGFβ1 immunostaining at the 1-week time point. At the 4-, 8-, and 12-week time points, disks in group A exhibited significantly increased proteoglycan and collagen type II synthesis compared with the other 2 groups (P<0.01). Abundant green fluorescence was observed in the disks in group B at the 12-week time point.
Conclusions:
Early degenerative NP cells are susceptible to AAV-mediated gene transfer in vitro and in vivo. The rapid and prolonged target protein expressions and increased matrix synthesis indicated that AAV-mediated therapeutic gene transfer can be a promising form of treatment for disk regeneration in vivo.
