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目的探讨错配修复基因-h MLH1启动子甲基化及基因失活在类风湿关节炎(RA)发生发展中的作用。方法选取2006年4月—2007年4月河北医科大学附属第二医院及第三医院新诊断的RA患者33例为病例组,另选择同期河北省血液中心提供的健康人群38例为对照组。取受试者外周血,以密度梯度离心法分离外周血单个核细胞。分别以RT-PCR法、蛋白印迹法检测外周血单个核细胞h MLH1 m RNA、蛋白相对表达量。经亚硫酸氢钠修饰后,分别设计h MLH1启动子甲基化和非甲基化的DNA引物,检测病例组和对照组患者h MLH1启动子甲基化结果。结果病例组h MLH1 m RNA相对表达量为0.56(0.24,0.69),低于对照组的0.80(0.68,0.93),差异有统计学意义(Z=-3.98,P<0.05)。病例组h MLH1蛋白相对表达量为0.70(0.62,0.76),低于对照组的0.80(0.68,0.94),差异有统计学意义(Z=-2.23,P<0.05)。病例组h MLH1启动子甲基化阳性率为90.9%(30/33),高于对照组的13.2%(5/38),差异有统计学意义(χ2=42.72,P<0.05)。h MLH1启动子甲基化程度与m RNA、蛋白相对表达量呈负相关(rs=-0.866、-0.541,P<0.01)。类风湿因子阳性患者h MLH1 m RNA、蛋白相对表达量低于阴性患者,差异有统计学意义(P<0.05);两者h MLH1启动子甲基化阳性率比较,差异无统计学意义(P>0.05)。结论 h MLH1启动子甲基化可能是h MLH1基因失活的重要机制,h MLH1异常高甲基化可能参与了RA的发生发展。
Objective To investigate the role of mismatch repair gene-h MLH1 promoter methylation and gene inactivation in the development of rheumatoid arthritis (RA). Methods From April 2006 to April 2007, 33 cases of newly diagnosed RA patients in the Second and Third Affiliated Hospital of Hebei Medical University were selected as the case group. Another 38 healthy subjects from Hebei Blood Center were selected as the control group. Peripheral blood mononuclear cells were isolated from peripheral blood mononuclear cells by density gradient centrifugation. The relative expression levels of hMLH1 mRNA and protein in peripheral blood mononuclear cells were detected by RT-PCR and Western blot respectively. After modified by sodium bisulfite, methylated and unmethylated DNA primers of hMLH1 promoter were designed respectively, and the methylation status of hMLH1 promoter in patients and controls was detected. Results The relative expression level of hMLH1 m RNA in case group was 0.56 (0.24,0.69), which was lower than that in control group (0.80,0.63). There was significant difference (Z = -3.98, P <0.05). The relative expression level of MLH1 protein in case group was 0.70 (0.62,0.76), which was lower than that in control group (0.80,0.64, P <0.05). The positive rate of hMLH1 promoter methylation in case group was 90.9% (30/33), which was higher than that in control group (13.2%, 5/38) (χ2 = 42.72, P <0.05). h MLH1 promoter methylation was negatively correlated with the expression of m RNA and protein (rs = -0.866, -0.541, P <0.01). The relative expression level of hMLH1 m RNA and protein in patients with rheumatoid factor positive was lower than that in patients with negative rheumatoid factor (P <0.05). There was no significant difference in the positive rate of hMLH1 promoter methylation > 0.05). Conclusion h MLH1 promoter methylation may be an important mechanism of hMLH1 gene inactivation. HHighly methylated MLH1 may be involved in the pathogenesis of RA.