骨保护素保护关节软骨细胞作用机制研究

Protective effect of osteoprotegerin on articular chondrocytes and its mechanism

目的探讨骨保护素(OPG)对间歇性循环牵张力(ICMT)加力诱导下退变的关节软骨细胞保护作用的机制。方法获取大鼠膝关节软骨细胞,传代至P2代进行实验,对细胞用FX-5000TM细胞体外力学刺激装置进行力学刺激,建立关节软骨细胞体外退变模型。P2代软骨细胞随机分为对照组、ICMT 3 d组(在与对照组相同培养条件下对细胞进行8%压强的ICMT,0.5 Hz,10 h/d,加力3 d)、OPG组(在细胞培养液中加入一定浓度的OPG培养细胞)、ICMT 3 d+OPG组(对用含有OPG的细胞培养液培养的细胞进行加力3 d)。培养3 d后收集各组细胞,应用倒置相差光学显微镜观察关节软骨细胞的表型变化,甲苯胺蓝染色法观察细胞表面形态,四噻唑蓝法检测不同浓度OPG和不同ICMT作用时间下关节软骨细胞存活率的变化。RT-PCR法检测各组关节软骨细胞中二型胶原、蛋白聚糖、SRY相关高迁移率族盒蛋白9(SOX-9)、基质金属蛋白酶13(MMP-13)mRNA表达水平,Western blot法检测各组关节软骨细胞中NF-κB信号通路相关蛋白MMP-13、p65、磷酸化p65(p-p65)蛋白表达水平。结果与对照组比较,ICMT 3 d组细胞发生梭形改变,细胞外基质减少;ICMT 3 d+20 ng OPG组细胞形态变化不明显,细胞外基质的丢失得到改善。ICMT 1 d组、ICMT 2 d组、ICMT 3 d组、5 ng OPG组、10 ng OPG组和20 ng OPG组与对照组关节软骨细胞存活率比较,差异均无统计学意义(均P>0.05)。与对照组比较,ICMT 1 d组、ICMT 3 d组细胞中二型胶原、蛋白聚糖、SOX-9 mRNA表达水平均下降,ICMT 3 d组细胞中MMP-13 mRNA表达水平升高,差异均有统计学意义(均P<0.05)。与ICMT 3 d组比较,ICMT 3 d+10 ng OPG组细胞中二型胶原、SOX-9 mRNA表达水平均升高,ICMT 3 d+20 ng OPG组细胞中二型胶原、蛋白聚糖、SOX-9 mRNA表达水平均升高,ICMT 3 d+10 ng OPG组、ICMT 3 d+20 ng OPG组细胞中MMP-13 mRNA表达水平均下降,差异均有统计学意义(均P<0.05)。与ICMT 3 d组比较,ICMT 3 d+5 ng OPG组、ICMT 3 d+10 ng OPG组、ICMT 3 d+20 ng OPG组MMP-13蛋白表达水平均下降(均P<0.05)。与ICMT 3 d组比较,ICMT 3 d+10 ng OPG组、ICMT 3 d+20 ng OPG组中p-p65蛋白表达水平均下降(均P<0.05)。结论OPG通过影响NF-κB信号通路可以抑制大鼠膝关节软骨细胞的退变,从而起到保护关节软骨的作用。

Objective To investigate the protective effects of the osteoprotegerin(OPG)on the articular chon-drocytes degeneration induced by intermittent cyclic mechanical tension(ICMT).Methods Rat articular chondrocytes were isolated and cultured in vitro.Knee articular chondrocyteswere isolated under aseptic conditions,and passaged to P2 generation chondrocytes for experiment.The chondrocytes were subject to ICMT with FX-5000TM cell strain loading system to establish cell degeneration model.The cells were divided into control group,ICMT 3 d group,OPG group and ICMT 3 d+OPG group.The changes of cellular morphology were observed by inverted phase contrast microscope,the phenotype of articular chondrocytes was identified by toluidine blue staining.The MMT was used to detect the changes in the survival rate of articular chondrocytes under different concentrations of OPG and different ICMT times.Type II collagen,aggrecan,SOX-9 and MMP-13 genes were detected by TR-PCR.Western blot was used to detect the expression of p-p65,p65 proteins in the nucleus and cytoplasm of articular chondrocytes in each group,and the expression of NF-κB signaling pathway-related MMP-13 was detected.Results Teluidine blue staining showed that the extracellular matrix of articular cartilage cells was reduced under the ICMT,which was improved in the OPG treatment group.Compared with the control group,no apoptosis was observed in the ICMT 1 d,ICMT 2 d,ICMT 3 d groups.Compared with the control group,the cell viability test showed that OPG did not cause apoptosis.Compared with the control group,the expression of type II collagen gene,aggrecan gene and SOX-9 gene in the ICMT 3 d group was decreased(all P<0.05),while the MMP-13 gene was increased(P<0.05).Compared with the ICMT 3 d group,the expression of MMP-13 in the ICMT 3 d+20 ng OPG group was decreased(P<0.05),the expression of type II collagen gene,aggrecan gene and SOX-9 gene was increased(all P<0.05).Compared with ICMT 3 d group,the p-p65 in the nucleus of ICMT 3 d+OPG group was decreased,the expression of MMP-13 protein in the NF-κB signaling pathway was also decreased(all P<0.05).Conclusion OPG can inhibit the degeneration of chondrocytes of articular cartilage by affecting NF-κB signaling pathway,so it can play a protective role in the knee joint and the articular cartilage.