5-氮杂胞苷与雌二醇对SLE患者CpG基序甲基化状态与DNMT1表达的影响

Effects of 5-azacytidine and estradiol on the methylation of CpG motifs and expression of DNA methyltransferase 1 in patients with SLE

目的 探讨5-氮杂胞苷与雌二醇对SLE患者与正常人CpG基序甲基化状态与DNA甲基转移酶1（DNMT1）表达的影响.方法 分离12例SLE患者与11例正常人外周血淋巴细胞.经植物血凝素刺激1天后,分为未处理组、5-氮杂胞苷组、雌二醇组.继续培养3d后分别用5-甲基胞嘧啶抗体与流式细胞仪检测CpG基序甲基化水平和实时定量逆转录-聚合酶链反应（RT-PCR）分析DNMT1的mRNA表达水平.结果 与非处理组相比,5-氮杂胞苷组SLE患者（t=18.60,P〈0.01）与正常人（t=5.63,P〈0.01）的CpG基序甲基化水平均下降.DNMT1表达水平（正常人t=2.17,P〉0.05,SLE患者t=1.56,P〉0.05）的下降无统计学意义.雌二醇组SLE患者与正常人的CpG基序甲基化水平（正常人t=1.93.P〉0.05;SLE患者t=1.53,P〉0.05）与DNMT1的mRNA（正常人t=0.11,P〉0.05;SLE患者t=0.93,P〉0.05）表达水平的差异均无统计学意义.结论 5-氮杂胞苷可抑制CpG基序甲基化状态,其抑制作用的产生与DNMT1表达水平的下降无明显相关性.雌二醇对CpG基序甲基化状态与DNMT1的表达水平的影响无统计学意义.

Objective To investigate the effects of 5-azacytidine and estradiol on the methylation of CpG motifs and expression of DNA methyltransferasel （DNMT1） mRNA in patients with systemic lupus erythematosus （SLE） and normal controls.Methods Peripheral blood lymphocytes isolated from 12 patients with SLE and 11 normal human controls were stimulated with phytohaemagglutinin for one day followed by additional 3-day culture with or without the presence of 5-azacytidine of 1 μmol/L or estradiol of 30μg/L respectively.Then.the methylation of CpG motifs was detected by flow cytometry using anti-5-methylcytosine antibody,and DNMT1 mRNA expression by real time reverse transcription-PCR Results After treatment with 5-azacytidine,a decrease wag observed in the methylation of CpG motifs, but not in the expression of DNMT1 mRNA in peripheral lymphocytes from patients with SLE （1=18.60,P〈0.01;t=1.56.P〉0.05） and in those from the normal controls （t=5.63,P〈0.01;t=2.17,P〉0.05） compared with untreated lymphocytes.Nevertheless,there were no significant changes in the methylation of CpG motifs or expression of DNMT1 mRNA in lymphocytes from patients with SLE （t=1.53,0.93,respectively,both P〉0.05） and normal controls （t=1.93,0.11,respectively,both P〉0.05） after the treatment with estradiol.Conclusions The methylation of CpG motifs is suppressed efficiently by 5-azacytidine,and the suppression is unlikely to be associated with the decrease of DNMT1 mRNA.Estradiol has no significant impact on the methylation of CpG motifs and expression of DNMT1 mRNA in lymphocytes.