目的应用一种 FOXP3 TSDR DNA去甲基化荧光定量PCR技术，并分析该方法在继发性肺结核患者治疗初期变化情况。方法选取2014年6月～2015年5月来深圳市龙华新区慢性病防治中心就诊的继发性肺结核病人47例作为研究组，健康对照40例，分离外周血单个核细胞，筛选 CD4＋CD25＋T细胞，提取基因组 DNA。利用去甲基化 FOXP3 TSDR特异性引物扩增对照组基因组DNA，通过酶切、克隆和回收纯化等过程构建质粒标准品，优化 FOXP3 TSDR去甲基化实时定量PCR技术，建立CD4＋CD25＋T细胞比例与 FOXP3 TSDR PCR 拷贝换算关系，用该方法分析对照组和研究组0周及治疗后2，4和8周时的调节T细胞频率（FOXP3 TSDR去甲基化），应用 spss16.0软件统计分析实验所得数据。结果0周时47例研究组抗酸染色均为阳性，对照组均为阴性。以抗酸染色结果分组：对照组、抗酸（1＋）组、抗酸（2＋）组、抗酸（3＋）组和抗酸（4＋）组其调节 T细胞频率分别为1.63％±0.70％，1.96％±0.10％，1.32％±0.32％，0.86％±0.21％和0.53％±0.12％，抗酸（2＋）组与对照组差异无统计学意义，抗酸（3＋）组与对照组差异有统计学意义。研究组0，2，4和8周时调节T细胞频率均值、范围及95％可信区间分别为1.05％（0.32％～2.03％），CI（0.93％，1.18％）；2.04％（0.95％～3.95％），CI（1.85％，2.24％）；3.44％（2.35％～4.95％），CI（3.27％，3.61％）；2.79％，（1.02％～4.27％），CI （2.60％，2.98％）。对照组与研究组0周时人群调节T细胞频率之间差异有统计学意义（t＝4.669，P＜0.05）。单变量方差分析显示治疗时间对研究组外周血调节T细胞频率（FOXP3 TSDR 去甲基化）水平变化有影响（F＝347.2，P＜0.001， df＝3，组内F＝407.4，P＜0.001，df＝3），治疗时间和分组交互效应呈线性关系（F＝678.2，P＜0.001，df＝1）。结论该方法敏感、特异，可以用于继发性肺结核患者的疗效监测。%Objective To analyze changes of DNA demethylation analysis of FOXP3 TSDR at the beginning of the secondary pulmonary tuberculosis patientsby utilizing real-time PCR technology.Methods To select 47 patients of secondary pulmona-ry tuberculosis as a research group from June 2014 to May 2015 and 40 healthy donors as a control group.The peripheral blood mononuclear cells (PBMC)of research group and control group were isolated.CD4+CD25+T cells were isolated from PBMC.Genomic DNA was isolated from CD4+CD25+T cells.PCR was performed in a final reaction volume containing dem-ethylation-specific primers.Plasmid standard was generated by PCR products were enzyme digestion,TOPO TA cloning,and recycling and purification.A real-time PCR system was established by quantitatively analyzing the specificity of FOXP3 TS-DR demethylation to treg (regulatory T-cell).Treg numbers of control group at week 0 and research group treated at week 0,week 2,week 4 and week 8 by using real-time PCR assay of the FOXP3 TSDR demethylation.The experimental data was analyzed by using SPSS 1 6.0 software.Results The M.tuberculosis in sputum of research group were positive by smear mi-croscopy,however the results of control group were negative.The treg frequency of control group,2+ group and 3+ group respectively was 1.63%±0.70%,1.96%±0.10% and 0.86%±0.21%,respectively.The difference between the treg fre-quency of control group and that of 2+ group by smear microscopy had not statistical significance,however which of 3+group was opposite.The average treg frequency of research group treated at week 0,2,4 and 8 respectively was at 1.05%, 2.04%,3.44% and 2.79%,range of which respectively was 0.32%~2.03%,0.95%~3.95%,2.35%~4.95% and 1.02%~4.27%,95% confidence interval of which respectively was (0.93%,1.18%),(1.85%,2.24%),(3.27%,3.61%) and (2.60%,2.98%).The treg frequency of difference between control group and research group at week 0 had statistical significance (t=4.669,P<0.05).The treg frequency was influenced by time of therapy,using One-Way ANOVA analysis (F=347.2,P<0.001,df=3,within-subjects Contrasts:F=407.4,P<0.001,df=3).Test of the treatment time and group interaction effect was linear (F=678.2,P<0.001,df=1).Conclusion DNA demethylation analysis of FOXP3 TSDR was high sensitivity and specificity in monitoring changes of treg at the beginning of the secondary pulmonary tuberculosis pa-tients.
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