Regulatory Effect Of Beijing Genotypes Of Mycobacterium Tuberculosis On T Lymphocyte Differentiation

OBJECTIVE: In this study,the Beijing genotype M.tuberculosis clinical strain lipoarabinomannan(BJ-LAM)was used as the research object,and co-cultured with human peripheral blood mononuclear cells by BJ-LAM,and then the cytokines in the supernatant were detected.This experiment explored the regulation of BJ-LAM on the differentiation of T lymphocytes,and also provided a reliable theoretical reference for the further application of BJ-LAM.Method:Screening of strains: On June 20,2018,a clinical isolate strain of Mycobacterium tuberculosis(WH322636)was screened from a sputum specimen of a tuberculosis patient in Wuhan Medical Treatment Center,Mycobacterium specific primers ITS and the RD105 region deletion method was used for Mycobacterium tuberculosis Mycobacterial Genotyping.The results showed that the clinical strain WH322636 was a slow-growing and acid-resistant Beijing genotype M.tuberculosis.Extraction and purification of lipoarabinomannan: Triton X-114 was used to extract clinical isolates WH322636,M.smegmatis mc2155,M.bovis BCG,M.tuberculosis H37 Ra crude extract,The crude extract extract was purified by a gel column.The obtained lipoarabinomannan(LAM)was developed by polyacrylamide gel electrophoresis combined with silver nitrate staining to determine the size of the four lipid arabinomannan structures.The effect of Beijing genotype M.tuberculosis LAM on T lymphocyte immune function: 30 cases of tuberculosis patients(including 19 smear negative TB patients)in Wuhan Medical Treatment Center were screened,and 12 healthy people were used as controls.2 ml of venous blood of the subject was taken,PBMC was extracted,and each participant's PBMC cells were stimulated with purified LAM(BCG-LAM,BJ-LAM).After 48 hours,PBMC released cytokines levels(IL-6,TNF?,IL2,IFN?,IL17,IL-10).Result:The clinical strain WH322636 is a slow-growing and acid-resistant Beijing genotype M.tuberculosis.Silver staining identification The molecular weight of LAM from small to large is M.smegmatis mc2155,Mycobacterium tuberculosis H37 Ra,Beijing genotype M.tuberculosis(WH322636),M.bovis BCG.After stimulation with BJ-LAM,the amount of IL-17 released from the supernatant of PBMCs was 41.73±4.32pg/m L,while the release of IL-17 from the supernatant of healthy control group was 21.50±4.566pg/m L,the difference was significant(P<0.005);the amount of IFN-? released from the supernatant of the PBMCs in the healthy group was 15.73±1.27pg/m L,and the release of IFN-? in the supernatant of the tuberculosis group was 31.41±3.642.pg/m L,the difference was significant(P<0.005);the amount of IL-10 released from the supernatant of the PBMCs in the healthy group was 0.6644±0.1012pg/m L,while the content of IL-10 in the supernatant of the tuberculosis group was 2.601±0.3952.Pg/m L,the difference was significant(P<0.01).Conclusion:IL-17 mediates the pro-inflammatory response,promotes the secretion of chemokines and neutrophil aggregation.The presence of IL-17 also facilitates the recruitment of protective cells,and the tissue-induced cell population accumulates at the site of infection.The release of IL-17 from the supernatant of PBMCs in the healthy group was higher than that in the blank control group,which may indicate that BJ-LAM has the ability to regulate the release of IL-17 by T cells and protect the body.IFN-? can regulate the expression levels of 30 genes,and has anti-bacterial infection and other immune regulation effects.BJ-LAM stimulates the whole blood ?-interferon to increase significantly,indicating that BJ-LAM has immunopotentiating effect on the body,showing its The diagnosis of bacillary tuberculosis has greater clinical value.IL-10 is an anti-inflammatory factor that plays a role in down-regulating the inflammatory response during bacterial infection and antagonizing inflammatory mediators.The release level of IL10 in tuberculosis group was higher than that in healthy group,suggesting that it can lead to down-regulation of mononuclear macrophage co-stimulatory molecules,resulting in decreased antigen presentation pathway and decreased activity of CD4+T and CD8+T.The level of IL-6 release from BJ-LAM-stimulated patients decreased slightly compared with the blank control group,which may indicate that the possibility of polarization to Th2 after BJ-LAM stimulation is not significant,but further data validation is needed.The results show that BJ-LAM has potential application value in diagnosis,immunotherapy and prevention,and also provides a reference for further study of its immune mechanism.