[1]武学成,冀红霞,魏玉娥.继发性肺结核患者治疗初期 FOXP3 TSDR DNA去甲基化变化情况分析[J].现代检验医学杂志,2016,31(03):84-87.[doi:10.3969/j.issn.1671-7414.2016.03.023]
　WU Xue-cheng,JI Hong-xia,WEI Yu-e.Situation Changes of DNA Demethylation Analysis of FOXP3 TSDR at the Beginning of Patients with Secondary Pulmonary Tuberculosis[J].Journal of Modern Laboratory Medicine,2016,31(03):84-87.[doi:10.3969/j.issn.1671-7414.2016.03.023]
点击复制

继发性肺结核患者治疗初期 FOXP3 TSDR DNA去甲基化变化情况分析()
分享到：

《现代检验医学杂志》[ISSN:/CN:]

卷:
第31卷
期数:
2016年03期
页码:
84-87
栏目:
论著
出版日期:
2016-06-15

文章信息/Info

Title:
Situation Changes of DNA Demethylation Analysis of FOXP3 TSDR at the Beginning of Patients with Secondary Pulmonary Tuberculosis
文章编号:
1671-7414(2016)03-084-05
作者:
武学成1; 冀红霞2; 魏玉娥3
1.深圳市龙华新区慢性病防治中心,广东深圳 518110;
2.深圳市龙华新区疾病预防控制中心,广东深圳 518110;
3.深圳市龙华新区人民医院检验科,广东深圳 518110
Author(s):
WU Xue-cheng1; JI Hong-xia2; WEI Yu-e3
1.Shenzhen Longhua New District Chronic Disease Prevention andControl Center, Guangdong Shenzhen 518110,China;
2.Shenzhen Longhua New District Centerfor Disease Control and Prevention,Guangdong Shenzhen 518110,China;
3.Department ofClinical Labo
关键词:
继发性肺结核;  调节T细胞去甲基化区域(treg-specific demethylated region FOXP3 TSDR);  叉头状/翅膀状螺旋转录因子(forkhead box P3 protein; Foxp3)
分类号:
R521.5; R392.11
DOI:
10.3969/j.issn.1671-7414.2016.03.023
文献标志码:
A
摘要:
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。
Abstract:
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 pulmonary tuberculosis as a research groupfrom 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 containingdemethylation-specific primers.Plasmid standard was generated by PCR productswere enzyme digestion,TOPO TA cloning,and recycling and purification.A real-time PCR system was established by quantitatively analyzing the specificity of FOXP3 TSDR 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 byusing real-time PCR assay of the FOXP3 TSDR demethylation.The experimental data was analyzed by using SPSS 16.0 software.Results TheM.tuberculosis in sputum of research group were positive by smear microscopy,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 frequency of control group and that of 2+ group by smear microscopy had not statistical significance,howeverwhich 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 TSDRwas high sensitivity and specificity in monitoring changes of treg at the beginning of the secondary pulmonary tuberculosis patients.

参考文献/References:

[1] World Health Organization.Global tuberculosis report 2014[M].Geneva:WHO Library Cataloguing-in-Publication Data,2014:08.
[2] Ohkura N,Kitagawa Y,Sakaguchi S.Development and maintenance of regulatory T cells[J].Immunity,2013,38(3):414-423.
[3] 钱 晨.结核感染T细胞酶联免疫斑点试验与结核抗体试验在诊断骨关节结核中的临床应用价值[J].现代检验医学杂志,2015,30(4):74-76. Qian C.Clinical value of tuberculosis infection T cell ELISPOT andTB antibody test in experimental tuberculosis of bones and joints[J].Journalof Modern Laboratory Medicine,2015,30(4):74-76.
[4] 赵英伦,胡溪源,李翠萍,等.肺结核患者血清caspase-3活性研究[J].现代检验医学杂志,2014,29(5):40-41,43. Zhao YL,Hu XY,Li CP,et al.Serum caspase-3 activity in tuberculosis patients[J].Journal of Modern Laboratory Medicine,2014,29(5):40-41,43.
[5] Ngalamika O, Liang G, Zhao M,et al.Peripheral whole blood FOXP3 TSDRmethylation:a potential marker in severity assessment of autoimmune diseases and chronic infections[J].Immunol Invest,2015,44(2):126-136.
[6] 张贺秋,赵雁林.现代结核病诊断技术[M].北京:人民卫生出版社,2013:47-55. Zhang HQ,Zhao YL.Modern TB diagnosis techniques[M].Beijing:People's Medical Publishing House,2013:47-55.
[7] Wieczorek G,Asemissen A,Model F,et al.Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue[J].Cancer Res,2009,69(2):599-608.
[8] Sakaguchi S,Setoguchi R,Yagi H,et al.Naturally arising Foxp3-expressing CD25+CD4+regulatory T cells in self-tolerance and autoimmune disease[J].Curr Top Microbiol Immunol,2006(305):51-66.
[9] Plesa G,Zheng L,Medvec A,et al.TCR affinity and specificity requirements for human regulatory T-cell function[J].Blood,2012,119(15):3420-3430.
[10] Katoh H,Zheng P,Liu Y.FOXP3:genetic and epigenetic implications for autoimmunity[J].J Autoimmun,2013(41):72-78.
[11] Littman DR,Rudensky AY.Th17 and regulatory T cells in mediating and restraining inflammation[J].Cell,2010,140(6):845-858.
[12] Lal G,Bromberg JS.Epigenetic mechanisms of regulation of Foxp3 expressing[J].Blood,2009,114(18):3727-3735.
[13] Hori S.Lineage stability and phenotypic plasticity of Foxp3+regulatoryT cells[J].Immunol Rev,2014,259(1):159-172.
[14] Ohkura N,Hamaguchi M,Morikawa H,et al.T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementaryevents required for Treg cell development[J].Immunity,2012,37(5):785-799.
[15] Lopez-Pastrana J,Shao Y,Chernaya V,et al.Epigenetic enzymes are thetherapeutic targets for CD4(+)CD25(+/high)Foxp3(+)regulatory T cells[J].Transl Res,2015,65(1):221-240.

备注/Memo

备注/Memo:
基金项目:2015年深圳市科技研发资金项目第二批基础研究项目(JCYJ20150325103202291)。 作者简介:武学成(1980-),男,硕士,副主任技师,医师,研究方向:细菌分子生物学。
更新日期/Last Update: 2016-06-25