Objective To investigate the role of transforming growth factor β(TGF-β)in the regulation of the gene expression of matrix metalloproteinase 13(MMP-13)in the human hyaline chondrocytes. Methods The human hyaline chondrocytes harvested enzymatically and cultured in DMEM supplemented with 20% fetus calf serum were divided into 7 groups. Group 1 was used as a contol, and 1 ng/ml TGF-β(group 2), 10 ng/ml TGF-β(group 3), 100 ng/ml TGF-β(group 4), 1 ng/ml TGF-β＋10 ng/ml IL-1β(group 5), 10 ng/ml TGF-β＋10 ng/ml IL-1β(group 6),and 100 ng/ml TGF-β＋10 ng/ml IL1β(group 7) were given for 12-hour coculture. The MMP-13 mRNA levels of passaged human hyaline chondrocytes were assessed by reverse transcriptionpolymerase chain reaction(RT-PCR) and real-time fluorescent quantitative PCR. Results TGF-β can increase the MMP-13 mRNA level respectively in the passagedhyaline chondrocytes. In the multifactor treated groups, TGF-β can decrease the MMP-13 mRNA level respectively and there was significant difference between groups (Plt;0.05).The level of MMP-13 mRNA expression had significant coherence withthe dosage of TGF-β. Conclusion The above results show that human chondrocytes express MMP-13 mRNA. TGF-β could cause a dosedependent stimulation on MMP-13 gene expression in human chondrocytes and have a potent effect of antagonizing IL-1β in osteoarthritis. TGF-β may play a crucial role in the occurrence anddevelopment of osteoarthritis through regulating MMP-13.
ObjectiveTo investigate the role of osteopontin (OPN) on the expressions of matrix metalloproteinase 13 (MMP-13) mRNA and protein in human knee osteoarthritic chondrocytes and to find the optimal time and concentration for OPN treatment. MethodsChondrocytes were isolated from articular cartilage tissues of patients with primary osteoarthritis (OA) and cultured using one step digestive treatment with collagenase typeⅡ. The chondrocytes were then identified using immunohistochemistry of collagen typeⅡ. The first generation of chondrocytes were stimulated with OPN at a concentration of 1μg/mL for 0, 24, 48, and 72 hours, and with OPN at the concentrations of 0, 0.5, 1, 2, and 4μg/mL for 48 hours. The levels of MMP-13 mRNA and protein expressions were measured with real-time fluorescent quantitative PCR and Western blot. ResultsThe immunohistochemical staining showed that first generation of chondrocytes expressed collagen typeⅡ. Both MMP-13 mRNA and protein expression levels in OA chondrocytes increased significantly in the presence of OPN (1μg/ mL) and peaked at 48 hours after incubation, showing significant difference between different time points (P < 0.05). The MMP-13 mRNA expression level in OA chondrocytes at the OPN concentration of 1μg/mL was significantly higher than those at the other concentrations (P < 0.05), and the MMP-13 protein expression level at the OPN concentration of 1μg/mL was significantly higher than that at 0μg/mL (P < 0.05). MMP-13 protein expression level at the OPN concentrations of 0.5, 2, and 4μg/mL were significantly higher than that at 0μg/mL (P < 0.05). ConclusionOPN induces up-regulation of MMP-13 mRNA and protein expressions in human knee osteoarthritic chondrocytes in time-and dose-dependent manners. The optimal time and concentration for OPN treatment are 48 hours and 1μg/mL, respectively.