Research On Gene Expression Profiles Of ESAT-6 And CFP-10 Stimulated CD4~+T Lymphocytes From Tuberculosis Patients

Backgroud Mycobacterium tuberculosis(MTB)has always been a threat to humans health as a pathogen of tuberculosis.According to the WHO latest statistics,currently about 10.4 million individuals diagnosed with active tuberculosis every year,the annual death is about 1.4 million people,more than one-third of the world's population affected with Mycobacterium tuberculosis.At the same time,with HIV infection and multi-drug resistance cases increased,the prevention and treatment of tuberculosis is in grim situation.There is a great significance to find a rapid,accurate,sensitive,specific test methods for the prevention and treatment of tuberculosis.IGRA has been developed as a method for diagnosis of TB in recent years and findings from several studies showed its sensitivity and specificity.Two mycobacterium tuberculosis specific proteins ESAT-6 and CFP-10 are used in IGRA as diagnostic antigens,which are used to detect the release of target cells IFN-? amount,in order to determine the infection of the subjects.However,there are few studies on the mechanism of IFN-? release from CD4 + T lymphocytes and the specific differences in expression genes of IFN-? stimulated by ESAT-6 and CFP-10.Objective: To detect and compare the different gene expression profiles of ESAT-6 and CFP-10 stimulated CD4+ T lymphocytes from tuberculosis patients peripheral blood using whole genome expression microarray screening,and to identify the changes in cell signaling pathways in order to provide a basis for further exploring the pathogenesis of tuberculosis.Methods: 10 ml peripheral venous blood was collected from three first diagnosed tuberculosis patients.PBMC were isolated by density gradient centrifugation and stimulated with ESAT-6 and CFP-10 for 6 hours.Then,CD4+ T lymphocytes were obtained from ESAT-6 and CFP-10 stimulated PBMC by immunomagnetic technique.The differences in gene expression before and after stimulation were detected using whole genome expression microarray technology.Bioinformatics analyses were used to identify genes and real time RT-PCR was applied to verify microarray data.Results: 1.After ESAT-6 stimulation,it had 2297 differentially expressed genes.Among them 1071 were up-regulated and 1226 were down-regulated(>2 fold changes and P < 0.05).Seventeen signaling pathways were involved based on data and five of which got involved in tuberculosis IFN-? release,including JAK-STAT signaling pathway,Toll like receptor signaling pathway,NF-?B signaling pathway,MAPK signaling pathway,and PI3K-Akt signaling pathway.2.After CFP-10 stimulation,it had 2954 differentially expressed genes.Among them 1360 were up-regulated and 1594 were down-regulated(>2 fold changes and P < 0.05).Twenty-one signaling pathways were involved based on data and five of which got involved in tuberculosis IFN-? release,including JAK-STAT signaling pathway,Toll like receptor signaling pathway,T cell receptor signaling pathway,MAPK signaling pathway,and TGF-beta signaling pathway.3.The expression trend of 7 genes chosen for further verification was consistent with the q RT-PCR results.Conclusions: After ESAT-6 and CFP-10 stimulation,obvious changes of gene expression profiles were observed.Those differentially expressed genes play important roles in regulation of signaling pathways in CD4+ T lymphocytes tuberculosis IFN-? release.Therefore,this study provides a solid theoretical basis for further investigations on the molecular pathogenesis of tuberculosis.