氧化应激相关细胞衰老指标与骨关节炎的相关性探讨

Correlation between oxidative stress related cell senescence indicators and osteoarthritis

目的 通过检测软骨细胞衰老相关指标,探讨软骨细胞衰老与骨关节炎(OA)发病的相关性。方法 选取2018年1月—2020年12月南京鼓楼医院骨科需手术住院的OA患者及非OA患者各6例,分离培养膝关节软骨细胞,分为正常软骨细胞组(对照组)、OA软骨细胞组(OA组),选P2代细胞分别进行β-半乳糖苷酶染色检测,收集细胞行流式细胞仪检测细胞活性氧(ROS)水平、线粒体膜电位,定量PCR(qPCR)检测细胞P21、P53基因mRNA表达,采用Western blotting检测软骨细胞中P21、P53蛋白水平。结果 OA组软骨细胞β-半乳糖苷酶染色阳性率显著增高,OA组ROS水平[(24.72±2.40)%]较对照组[(4.96±0.14)%]明显升高(P<0.01),OA组JC-1染色绿色荧光强度[(21.87±3.03)%]即细胞线粒体膜电位下降明显高于对照组[(3.13±0.25)%,P<0.01];OA组P21、P53基因mRNA表达量(2.72±0.21、2.90±0.23),较对照组表达量(1.00±0.03、1.00±0.06)显著增加(均P<0.05)。OA组周期蛋白P21、P53表达量(0.51±0.02、0.55±0.03)亦较对照组表达量(0.40±0.03、0.45±0.02)显著增加(P<0.01)。结论 OA患者软骨细胞衰老较对照组显著,氧化应激诱导的细胞衰老可能在OA发病中起关键作用,抗氧化应激或可作为OA治疗的有效靶点。

Objective To investigate the correlation between chondrocyte senescence and osteoarthritis(OA) by detecting chondrocyte senescence related indicators.Methods The OA patients and non-OA patients hospitalized in the Department of Orthopedics in Nanjing Drum Tower Hospital were selected from January 2018 to December 2020.Normal knee chondrocytes and OA knee chondrocytes were isolated and cultured,and divided into normal human chondrocytes group(control group) and OA chondrocytes group(OA group).P2 generation cells were selected for the experiment.β galactosidase staining was performed.The reactive oxygen species(ROS) level and mitochondrial membrane potential were detected by flow cytometry.The mRNA expressions of P21 and P53 genes were detected by quantitative PCR(qPCR),and the protein levels of P21 and P53 in chondrocytes were detected by Western blotting.Results The positive rate of β-galactosidase staining in chondrocytes in the OA group was significantly higher than that in the control group.The level of ROS in the OA group [(24.72±2.40) %] was significantly higher than that in the control group [(4.96±0.14) %,P<0.01].The green fluorescence intensity of JC-1 staining in the OA group [(21.87±3.03) %] was significantly higher than that in the control group [(3.13±0.25) %,P<0.01].The mRNA expressions of P21 and P53 genes in the OA group were(2.72±0.21) and(2.90±0.23),which were significantly higher than those in the control group(1.00±0.03) and(1.00±0.06),all P<0.05.The expression levels of cyclin P21 and P53 in the OA group [(0.51±0.02) and(0.55±0.03)] were also significantly higher than those in the control group [(0.40±0.03) and(0.45±0.02),P<0.01].Conclusion Compared with normal chondrocytes,OA chondrocytes senesce significantly.Oxidative stress-induced cell senescence may play a key role in the pathogenesis of OA.Anti-oxidative stress may be an effective target for the treatment of OA.