骨桥蛋白对人膝骨关节炎软骨细胞基质金属蛋白酶13表达的影响

EFFECT OF OSTEOPONTIN ON EXPRESSION OF MATRIX METALLOPROTEINASE 13 IN HUMAN KNEE OSTEOARTHRITIC CHONDROCYTES

目的探讨骨桥蛋白(osteopontin,OPN)对人膝骨关节炎软骨细胞基质金属蛋白酶13(matrix metall oproteinase 13,MMP-13)表达的影响,并确定OPN干预的最佳时间和浓度。方法采用原发膝骨关节炎患者自愿捐赠的膝关节软骨,Ⅱ型胶原酶一步消化法分离培养软骨细胞,并行Ⅱ型胶原免疫组织化学染色鉴定。取第1代软骨细胞,分别以1μg/mL浓度的OPN干预培养0、24、48、72 h,以及0、0.5、1、2、4μg/mL浓度的OPN干预培养48 h;实时荧光定量PCR和Western blot检测MMP-13 mRNA和蛋白表达水平。结果免疫组织化学染色鉴定示Ⅱ型胶原染色呈阳性。经1μg/mL OPN干预培养后,软骨细胞MMP-13 mRNA及蛋白表达水平均呈升高趋势,48 h时达峰值,各时间点间比较差异均有统计学意义(P<0.05)。不同浓度OPN干预培养48 h后,1μg/mL组MMP-13 mRNA表达水平较其余组显著上升(P<0.05),蛋白表达水平亦显著升高,但仅与0μg/mL比较差异有统计学意义(P<0.05);0.5、2、4μg/mL组仅MMP-13蛋白较0μg/mL组显著上升,差异有统计学意义(P<0.05)。结论 OPN可明显上调人膝骨关节炎软骨细胞MMP-13表达,且具备时间和浓度依赖性,干预最佳时间及浓度分别为48 h及1μg/mL。

Objective To 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. Methods Chondrocytes were isolated from articular cartilage tissues of patients with primary osteoarthritis （OA） and cultured using one step digestive treatment with collagenase type II. The chondrocytes were then identified using immunohistochemistry of collagen type II. The first generation of chondrocytes were stimulated with OPN at a concentration of 1 μg/mL for 0, 24, 48, and 72 hours, and with OPN at the concentrations of 0, 0.5, 1, 2, and 4 μg/mL for 48 hours. The levels of MMP-13 mRNA and protein expressions were measured with real-time fluorescent quantitative PCR and lWestern blot. Results The immunohistochemical staining showed that first generation of chondrocytes expressed collagen type II. Both MMP-13 mRNA and protein expression levels in OA chondrocytes increased significantly in the presence of OPN （1 μg/ 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 1 μg/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 1 μg/mL was significantly higher than that at 0 μg/mL （P〈0.05）. MMP-13 protein expression level at the OPN concentrations of 0.5, 2,and 4 μg/mL were significantly higher than that at 0 μg/mL （P〈0.05）. Conclusion OPN 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 1 μg/mL, respectively.