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Clinical Observation On T-cell Exhaustion Of Tuberculosis And Research On Its Animal Model Treatment

Posted on:2018-09-06Degree:MasterType:Thesis
Country:ChinaCandidate:C GanFull Text:PDF
GTID:2334330533957750Subject:biology
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Globally,the infection and death rates of tuberculosis are still high.In some development country like China and India,it gives much pain to the society and people.At the beginning of the Mycobacterium tuberculosis(M.tuberculosis)infection,90% of people can clear the bacteria by own immunity.Finally,10% of people develop active TB disease during their lifetime.In the long stage of TB infection,it weakened the immune system and the bacteria revived which made the disease became to chronic infection.In the persistent stimulation of MTB infection,the immunity of the patients always had poor immunity.How could the MTB weaken the immunity? Did the T cell become exhausted? We detected the T cell exhaustion indicators of different TB patients to study the mechanism of T cell exhaustion.Objective The purpose of the current study is to explore whether T-cell dysfunction and exhaustion would happen in cavitary tuberculosis(TB)patients with a large of Mycobacterium tuberculosis(M.tuberculosis)infection.Methods The 51 cases of tuberculosis patients including 11 sputum smear-positive cavitary TB patients from Lanzhou Pulmonary Hospital and 16 healthy candidates were enrolled.1,The peripheral blood mononuclear cells(PBMCs)were separated by lymphocyteisolation.2,IFN-γ secretion by lymphocytes was tested by ELISPOT.3,Interleukin-2(IL-2)secretion was tested by ELISA.3,T cell proliferation with the stimulation of specific antigens was analyzed by flow cytometry(FCM).4,T cell immunoglobulin mucin domain containingmolecules 3(TIM-3)expressed on CD4+ and CD8+ T cell surface was also detected by flow cytometry.5,the numbers of CD4+,CD8+T cells and CD4+/CD8+TIM-3+T cells were calculated.6,We checked the medical clinical records of patients to acquire the results of red blood cell(RBC),blood platelet(PLT),white blood cell(WBC),hemoglobin(Hb)and mononuclear cell(MON).7,we analyzed the clinical material about sex,age,course of disease,untreated or retreated,sputum smear,cavitary TB or not.It would help us to understand what kind of patients might have T cell exhaustion.Results In the previous work of our lab,we found the correlative factors of T cell exhaustion were cavitary TB,sputum smear and course of disease.The 51 cases of TB patients and 16 cases of health donors were divided into 4 groups: HD group,N-TB group,PNC-TB group,PC-TB group.The results demonstrated that comparing with the healthy control the number of IFN-γ secreting lymphocytes from sputum acid-fast stain(AF)negative(-)non-cavitary TB patients and AF positive(+)non-cavitary TB patients increased,and the proliferation of T cells in sputum AF(-)TB patients increased.For sputum AF(+)cavitary TB patients,the abilities of T cells secreting IFN-γ and proliferation were significantly reduced comparing with the other two groups of TB patients.All of the TB patients had lower IL-2 secretion comparing with the HD group.TIM-3 expression increased in all TB patients but there is no difference among them.Conclusion 1,TIM-3 expression play a role in the whole process of TB disease;2,The IL-2 secretion of T cells began to reduce in the early stage of TB disease;3,T cell exhaustion correlated with cavitary TB,sputum smear and course of disease;4,The Hematopoietic stem cell of PC-TB patients might be exhausted.5,the function of T lymphocytes in the sputum AF(+)cavitary TB patients decreased,which suggested that a large of M.tuberculosis persistent infection would lead to exhaustion of T lymphocyte.Tuberculosis is a kind of chronic infectious disease caused by mycobacterium tuberculosis(M.tuberculosis).M.tuberculosis can not only cause intrapulmonary tuberculosis but although extrapulmonary tuberculosis.It exists for a long time in the body impairing the body’s immune function.Like HBV、HCV、HIV infection,these viral disease caused by persistent infection made T cells exhausted,T cell exhaustion caused by M.tuberculosis persistent infection got more and more attentions by researchers these years.Exhausted T cells show a state of up-regulation the expression of inhibitory receptors,reduction of proliferation,loss of cytotoxic activity and reduction of cytokine secretion.The T cell exhaustion once occured,the TB patients immunity became very weak,disease continue to deteriorate and difficult to recover.To overcome T cell exhaustion in tuberculosis,restore the immunity of TB patients,animal model was constructed to verify the two drugs reversing T cell exhaustion in this study.Objective The purpose of this study is to explore the mechanism of T cell exhaustion induced by persistent Mycobacterium Tuberculosis Antigens.To evaluate effects of Bushen Jianpi Recipe(BSJP)and metformin on reversion of T cell exhaustion in mice.Methods The 3 weeks of C57BL/6 mice were divided into 5 groups randomly: na?ve group,transient group,untreated group,BSJP group and metformin group.In the 1st week,animals were primed with BCG(5×106 CFU).In the 6th week,animals were boosted with 20μg of Mycobacterium bovis BCG Purified Protein Derivative(PPD)once for a week and lasted for 16 times to induce T cell exhaustion that happened in late stage of human tuberculosis.In 24 th week,BSJP was used to treat the immune exhausted mice for 3 weeks.From 7th week to 26 th week,metformin was given to metformin group in the drinking water.After model construction,some immunodetections were performed as below: Flow Cytometry was used to detect the PD-1 expression on CD4+ and CD8+ T cells,the numbers of CD4+ and CD8+T cells,the number of hematopoietic stem cell;ELISA was used to detect IL-2 and IFN-γ secretion;Bacteria Count in lung tissue following BCG infection was used to detect the protective immune efficacy in mice models.Results Compared with the transient antigen stimulation group,the persistent antigen stimulation group had lower level of IFN-γ and IL-2 secretions(p<0.01);the numbers of CD4+ and CD8+T cells decreased(p<0.01)and the level of inhibitory receptor PD-1 got higher(p<0.01);the protective efficacy against BCG challenging decreased(p<0.01).With the treatment of BSJP and metformin,the production of cytokines IFN-γ and IL-2 increased(p<0.01);the numbers of CD4+ and CD8+T cells increased and PD-1 expression level on CD4+T cells decreased(p<0.01);the protective efficacy against BCG challenge increased(p<0.01).The results showed that BSJP and metformin can reduce the PD-1 expression,improve the IL-2 and IFN-γ secretion,and increase the protection against BCG challenge.Conclusion This study reveals that Bushen Jianpi Recipe can reverse T cell exhaustion induced by persistent tuberculosis antigen stimulation in mice,which gives a hope to overcome T cell exhaustion with Chinese herbal in clinic.Metformin can stop the progress of T cell exhaustion in mice model.
Keywords/Search Tags:Tuberculosis, cavitary tuberculosis, T cell exhaustion, Bushen Jianpi Recipe, Metformin, Antigen persistent stimulation
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