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目的明确肺结核病人DNA低甲基化状态与TETs、TDG之间的相关性。方法在前期研究结果的基础上,收集健康对照和活动性肺结核病人治疗前全血RNA,通过实时荧光定量PCR(Real-time PCR)方法,对参与DNA甲基化下调的DNA去甲基化酶,即十-十一染色体异位酶基因(TETs,包括TET1、TET2、TET3)及胸腺嘧啶糖基酶(TDG)进行定量检测。利用H37Rv裂解抗原以及5-氮杂胞嘧啶核苷(5azac)刺激物刺激人肺癌细胞系A549和人支气管上皮细胞Beas-2B两种细胞系,并收集刺激后不同时间点细胞DNA、RNA。利用甲基化敏感性限制性分析(MSRA)对所收集DNA样本进行分析;进一步利用Real-time PCR(RT-PCR)方法对细胞RNA样本进行检测。结果在肺结核病人组中,DNA去甲基化酶表达均显著升高(P<0.05)。H37Rv抗原、5azac导致DNA甲基化水平降低,且刺激时间越长甲基化降低越明显。且在刺激第4天A549细胞中H37Rv刺激组DNA去甲基化酶TET2、TET3上调,而在Beas-2B中为H37Rv刺激组DNA去甲基化酶TDG上调,与临床检测结果相符。结论在活动性肺结核病人中及体外细胞刺激实验H37Rv抗原刺激组中,DNA甲基化呈现低甲基化趋势;DNA去甲基化酶可能是参与其DNA低甲基化趋势的主要的酶。
Objective To determine the relationship between DNA hypomethylation and TETs and TDG in patients with pulmonary tuberculosis. Methods Based on the results of the previous study, whole blood RNA of healthy controls and patients with active pulmonary tuberculosis before treatment were collected. Real-time quantitative PCR (Real-time PCR) was used to detect DNA demethylase (TETs, including TET1, TET2, TET3) and thymidine glycosylase (TDG). The human lung cancer cell line A549 and the human bronchial epithelial cell line Beas-2B were stimulated with H37Rv cleavage antigen and 5-azacitidine (5azac) stimulus, and the cellular DNA and RNA were collected at different time points after stimulation. The collected DNA samples were analyzed by restriction analysis of methylation sensitivity (MSRA) and the RNA samples were further detected by real-time PCR (RT-PCR). Results In patients with pulmonary tuberculosis, DNA demethylase expression was significantly increased (P <0.05). H37Rv antigen, 5azac lead to DNA methylation levels decreased, and the longer the stimulation of methylation decreased the more obvious. The DNA demethylase TET2 and TET3 were up-regulated in H37Rv-stimulated group in A549 cells on day 4, whereas the DNA demethylase TDG up-regulated in H37Rv-stimulated group in Beas-2B was consistent with the clinical test results. Conclusion DNA methylation shows a tendency of hypomethylation in active pulmonary tuberculosis patients and H37Rv antigen stimulation group in vitro. DNA demethylase may be the main enzyme involved in the DNA hypomethylation.