瘦素启动子甲基化在骨关节炎发病中的作用

The role of promoter CpG islands methylation of leptin gene in osteoarthritis

目的 探讨瘦素启动子甲基化在骨关节炎（OA）发病中的作用.方法 取OA组和对照组（创伤行截肢手术,除外OA等关节炎性疾病）患者膝骨关节标本,培养膝关节软骨细胞,采用不同浓度（5.0 μmol/L、10.0 μmol/L、20.0 μmol/L）不同时间（12 h、24 h、48 h、72 h、96h、120 h、168 h）5-氮杂-2＇-脱氧胞苷（5-Aza-CdR）刺激软骨细胞,实时定量PCR检测膝关节软骨细胞瘦素mRNA表达,同时使用Epityper DNA甲基化分析技术枪测该基因启动子部分区域（-280～＋79）的甲基化状态.结果 （1）10μmol/L 5-Aza-CdR刺激OA组软骨细胞,其瘦素mRNA表达水平增高,72 h最为显著.（2）OA组5-Aza-CdR刺激72 h后软骨细胞瘦素mRNA表达最高,对照组无5-Aza-CdR刺激软骨细胞瘦素mRNA表达最低.（3）使用非先导分层聚类分析法对瘦素启动子区域进行分析,2组甲基化模式存在差异,且 5-Aza-CdR刺激前后甲基化模式也不相同.结论 瘦素启动子部分位点的去甲基化可能是引起该基因异常表达导致OA发生的始动因素之一.

Objective To investigate the effects of 5-Aza-CdR（ methylation transferase inhibitor） on the expression levels of leptin gene in chondrocytes and methylation states of leptin promoter region between osteoarthritis （OA） group and control. Methods The chondrocytes in osteoarthritis group were treated with 5-Aza-CdR with different doses and time-points, and the expression level of leptin was detected by real-time polymerase chain reaction for picking up the optimum dose and time-point. Next, the chondrocytes in 5 osteoarthritis patients and 5 control patients （amputation due to severe trauma） were treated with 5-Aza-CdR. Lastly, leptin mRNA expression levels in the four groups osteoarthritis and control chondrocytes treated with/without 5-Aza-CdR were measured by real-time PCR and the methylation state of promoter region （ - 280- ＋ 79） was detected by epityper quantitative DNA methylation analysis. Results （ 1 ） After treating the chondrocytes in OA groups with 10 μmol/L 5-Aza-CdR for 72 h, the mRNA expression levels of leptin were increased significantly. （ 2 ） The mRNA expression levels of leptin were significantly different among the four groups （ P 〈 0. 05 ）, and the chondrocytes in osteoarthritis groups treated with 5-Aza-CdR showed a marked induction of leptin mRNA expression. （3） Analysis of quantitative methylation data using an unsupervised hierarchical clustering algorithm, showed that methylation patterns of leptin promoter was different between control and osteoarthritis chondrocyte treated with/without 5-Aza-CdR. Conclusion Demethylation of leptin promoter might up-regulate leptin gene expression level and it might contribute to osteoarthritis.