EXPRESSION OF HUMAN TRANSFORMING GROWTH FACTOR β1 GENE MEDIATED BY ADENOVIRUS IN HAMSTRING TENDON AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS

Abstract

To detect the expression of human transforming growth factor β1 (hTGF-β1) gene mediated by adenovirus (Ad) in hamstring tendon after anterior cruciate ligament (ACL) reconstruction in rabbits. Ad-hTGF-β1 and Ad-green fluorescent protein (GFP) were diluted to 5×108 PFU/mL with DMEM. Forty-eight New Zealand white rabbits were divided into 3 groups randomly (n=16) for ACL reconstruction with hamstring tendon autograft. Hamstring tendon was cultured and transfected with Ad-hTGF-β1 (group A) and Ad-GFP (group B) for 12 hours before ACL reconstruction, and was cultured with DMEM in group C. After 12 hours of transfection, green fluorescence was observed in groups A and B under fluorescence microscopy. At 2, 4, 6, and 8 weeks after operation, the hamstring tendon was harvested to detect the mRNA and protein expressions of hTGF-β1 by real time fluorescence quantitative PCR and Western blot. Green fluorescence was observed after 12 hours of transfection in groups A and B. TGF-β1 protein level reached (221.0±12.2) ng/mL at 12 hours in group A. The hTGF-β1 mRNA expression could be detected in group A, but it could not be detected in group B and group C. The mRNA expression levels of hTGF-β1 were 1.004±0.072 at 2 weeks, 0.785±0.038 at 4 weeks, 0.469±0.053 at 6 weeks, and 0.172±0.021 at 8 weeks in group A, showing significant difference (P<0.05). Western blot results showed weakly positive band in groups B and C; the protein expression of TGF-β1 in group A was significantly higher than that in groups B and C (P<0.05), but no significant difference was found between groups B and C P>0.05). The protein expression of TGF-β1 gradually reduced with time, showing significant difference between different time points (P<0.05). Ad-hTGF-β1 can transfect the hamstring tendon successfully, and it can effectively express for a long time after ACL reconstruction.