基质Gla蛋白MGP甲基化水平与溃疡性结肠炎患者免疫功能的相关性研究

Correlation between MGP methylation of matrix Gla protein and the immune function in patients with ulcerative colitis

目的研究基质Gla蛋白(MGP)在溃疡性结肠炎(ulcerative colitis,UC)中的表达和启动子区甲基化状态,探讨MGP对UC免疫平衡的影响。方法纳入UC患者105例,并收集健康者外周血为对照组,2组均被用于观察MGP的表达和甲基化变化。使用DSS构建小鼠UC模型,荧光定量PCR检测MGP的m RNA表达量;免疫组织化学和蛋白免疫印迹法检测中MGP的表达。基于甲基化敏感酶和甲基化依赖酶酶切、并结合荧光定量PCR方法分析UC组中MGP基因启动子区甲基化状态。构建DNMT-1过表达质粒(pcDNA3.1-DNMT-1),静脉注射UC小鼠中。ELISA检测人或动物的外周血中的炎症因子INF-γ、IL-2、IL-4、IL-10和免疫球蛋白-G/A/M(IgG/IgA/IgM)的水平。皮尔森法分析相关性。结果实时PCR证实,与对照组比,UC组的MGP在mRNA和蛋白水平上均过表达(P<0.05);免疫组织化学证实UC组织中MGP的阳性。UC组中MGP的启动子区CpG岛甲基化水平降低(均P<0.05),而且INF-γ、IL-4、IL-10和免疫球蛋白-G/A(IgG/IgA)均与MGP的甲基化水平显著相关(均P<0.05)。pcDNA3.1-DNMT-1组的INF-γ上调,且IL-10下调(均P<0.05)。结论UC中MGP上调或MGP启动子区甲基化下调介导UC的免疫失衡。

The study aimed to investigate the expression of matrix Gla protein(MGP)in ulcerative colitis(UC)and the methylation status of promoter region of MGP,and to explore the effect of MGP on UC immune balance.Total of 105 cases of UC patients were included,and peripheral blood of healthy individuals were collected as control group.The changes of MGP expression and methylation were observed in the two groups.DSS was used to construct a mouse UC model,and fluorescence quantitative PCR was used to detect MGP mRNA expression;immunohistochemistry and Western blot were used to detect MGP expression.The methylation status of the MGP gene promoter region in the UC group was analyzed based on the methylation-sensitive enzyme and methylationdependent enzyme digestion combined with fluorescent quantitative PCR.The DNMT-1 overexpression plasmid(pcDNA3.1-DNMT-1)was constructed and injected intravenously into UC mice.ELISA was used to detect the levels of inflammatory factors INF-γ,IL-2,IL-4,IL-10 and immunoglobulin-G/A/M(IgG/IgA/IgM)in human and animal peripheral blood.Correlation analysis was conducted by using Pearson method.Data showed that the MGP in the UC group was overexpressed at the mRNA and protein levels(P<0.05).Immunohistochemistry confirmed the positive of MGP in UC tissue.In the UC group,the methylation level of CpG island in the promoter region of MGP decreased(both P<0.05).Moreover,the levels of INF-γ,IL-4,IL-10 and immunoglobulin-G/A(IgG/IgA)were significantly correlated with the methylation level of MGP(all P<0.05).In pcDNA3.1-DNMT-1 group,INF-γwas up-regulated,while IL-10 was down-regulated(both P<0.05).In conclusion,up-regulation of MGP or down-regulation of MGP promoter methylation mediates UC immune imbalance.