肺结核中DNA低甲基化趋势与TETs及TDG的相关性研究

The correlation study of TETs,TDG and DNA low methylation trend in pulmonary tuberculosis

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 Show the correlation of the downregulated DNA methylation trend and Ten-eleven translocation enzymes （TETs） or thymine DNA glycosylase （TDG）. Methods In previous research, we found a downregulated DNA methylation trend in tuberculosis patients, so we firstly collect healthy controls and active tuberculosis patients＂ whole blood RNA to quantitatively detect four genes of DNA demethylases which could forwardly downregulate the DNA methylation level （mainly including TET1, TET2, TET3 and TDG） by means of Real-time PCR method. Subsequently we use H37Rv cracking antigen and positive stimulus （5azac） to stimulate A549, Beas-2b two cell lines to conduct the validation experiments in vitro. And then collect the stimulated cell DNA, RNA of different time points. Use the DNA samples to process methylation sensitive restriction analysis （MSRA） test in order to confirm the DNA methylation level after stimulation. And use the cell RNA samples to test the genes by the method of Real-time PCR as mentioned before. Results All the four genes appear upexpressed in the active tuberculosis patients （P〈0.05）. The result of MSRA shows the H37Rv antigen and 5-azac positive stimulus can cause DNA methylation level＇s drop as the genome DNA methylation chip shows, and it drops more as the time goes. And the DNA demethylases are increased as the time grows in the H37Rv-sti groups of both A549 and Beas-2B. And on the fourth day, TET2 and TET3 are differently up-regulation expressed in the H37Rv-sti group of A549 cellline. And TDG is differently increased in the H37Rv-sti group of Beas-2B cellline. Conclusion In the H37Rv antigen stimulation group and active tuberculosis patients, DNA methylation showed a trend of low methylation ; DNA demethylases was the main enzyme resulting in the DNA low methylation trend.