Situation Changes of DNA Demethylation Analysis of FOXP3 TSDR at the Beginning of Patients with Secondary Pulmonary Tuberculosis / 现代检验医学杂志

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 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 ContrastsF=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.