骨关节炎患者关节软骨TIMP-3启动子区甲基化水平的初步研究

目的探讨关节软骨中组织金属蛋白酶抑制因子-3（TIMP～3）基因启动子区甲基化与其蛋白表达的相关性，并分析TIMP一3基因CpG岛异常甲基化与骨关节炎（OA）的关联性。方法应用甲基化特异性的聚合酶链反应（MSP）技术和免疫组织化学SP法分别检测14例健康人的正常关节软骨细胞和35例骨关节炎（OA）患者软骨细胞TIMP-3基因启动子区甲基化和蛋白表达情况。结果OA患者和健康人关节软骨中TIMP-3基因启动子区均有甲基化修饰，其阳性率分别为74.3％（26／35）和35．7％（5／14），0A组TIMP-3基因启动子区甲基化率明显高于健康组（P〈0．05）。14例健康人中，软骨细胞TIMP-3蛋白表达阳性10例（71．4％），而35例OA患者中，软骨细胞TIMP-3蛋白表达阳性11例（31．4％）。24例OA患者软骨细胞蛋白表达阴性的标本中．TIMP-3启动子区甲基化阳性21例（87．5％）；26例TIMP-3启动子区甲基化阳性的标本中，蛋白表达阴性21例（80．8％），TIMP-3启动子区甲基化与蛋白表达呈显著负相关（P〈0．05）。结论启动子区CpG岛高甲基化是OA患者关节软骨细胞TIMP-3表达失活的主要机制之一，其可能参与了OA的发生和发展。

Investigation of TIMP-3 gene promoter methylation level in joint carti- lage of osteoarthriti patients

Objective To investigate the relationship between the methylation of tissue inhibitor of metalloproteinase-3 （TIMP-3） gene promoter and its protein expression, to study the potential role of hyper-methylation of TIMP-3 gene in the development of osteoarthritis. Methods The CpG islands methylation levels of TIMP-3 promoter in joint cartilage cells were studied by methylation-specific PCR（MSP）. TIMP-3 protein expression in joint cartilage cells were detected by immunohistochemical method in 35 osteoarthritis （OA） patients and 14 control health people. Results The positive percent of TIMP-3 promoter methylation levels in healthy people and OA patients were 35.7%（5/14） and 74.3%（26/35） respectively. The positive percent of TIMP-3 protein expression in OA patients were higher than healthy people （P〈 0.05）; In cartilage cells, the positive percent of TIMP-3 protein expression in healthy people and OA patients were 71.4%（10/14） and 31.4%（11/35） respectively. In 24 cases of OA samples with negative cartilage cells protein expres- sion, 21 cases （87.5%）was found with positive TIMP-3 promoter methylation, and in 26 cases of patients with positive TIMP-3 promoter methylation, protein expression was negative in 21 cases （80.8%）;Promoter methylation and TIMP-3 protein expression level showed significantly negative correlation. Conclusion TIMP-3 promoter is hyper-methlated in joint cartilage cells of OA patients. The hyper-methylation affects the protein expression of TIMP-3, which may play a crtical role in the occurrence and development of OA.