The Study On Regulation Of Macrophage Inflammatory Cytokines By BCG

Objective: Despite one hundred years of research,the infection of tuberculosis is still a major health problem worldwide,and it is the main cause of death.Ten million new cases are diagnosed each year,and 2 million people die from tuberculosis.The increased incidence of tuberculosis and AIDS related to the increase,and there have been a lot of drug-resistant Mycobacterium tuberculosis.Generally speaking,the host immune system can inhibit the growth of Mycobacterium,can lead to potential infection,is to survive in the granuloma but not active bacteria,potentially infected 1/3 of the world's population have tuberculosis,only ten percent people for the development of active pulmonary tuberculosis.Combined with Mycobacterium tuberculosis antigens on the surface of dendritic cells and macrophages and pattern recognition receptors leads to the activation of these cells and the secretion of cytokines.These cytokines can modulate the innate immune response,and can activate the acquired immunity.Cytokine production after Mycobacterium tuberculosis infection does not always play a protective role on the body,some are beneficial for tuberculosis.IL-23 belong to IL-12 family and is composed by two subunits(P40 and P19)consisting of two heterologous dimer structure,activation of antigen-presenting cells can produce IL-23,which plays an important role in maintaining the Th17 positive cells in vivo,there are correlation between Th17 cells with tumor and inflammation related diseases.In this study,we first observed the effect of BCG on the T cell subsets after inoculation,and then to study the pro-inflammatory and anti inflammatory cytokines produced by antigen presenting cells under BCG stimulation,and the regulation mechanism of BCG on IL-23 was mainly studied.Through the above research can lay the molecular basis for the clinical application.Method:1 Preparation and culture of mouse BMDM.2 The lymph node cells which from BCG inoculated C57BL/6 mice were collected and were detected T cell subsets.3 ELISA method was used to detect the amount of IL-1B,IL-6,IL-10,IL-23 and IL-12 in the supernatant of BMDM after stimulated by BCG.4 ELISA method was used to detect the level of IL-23 in the supernatant of RAW264.7 after stimulated by BCG.The expression of IL-23p19 m RNA was detected using Real time quantitative PCR.5 The random primers were designed for the DNA IL-23p19,changes in IL-23p19 before transcription was checked using real-time quantitative PCR in RAW cells after BCG stimulation.6 RAW264.7 cells were stimulated by BCG and add the transcription inhibitor dichlorobenzimidazole(DRB)+ ACD,to prevent the new RNA generation,p19 m RNA decay were detected in two groups at different time point.7 The total p65 and phosphorylated p65 were detected in RAW264.7 cells after stimulated by BCG using Western blotting method.8 RAW264.7 cells were treated with different concentrations of NFkappa B signal transduction pathway blocking agent SN50,p19 m RNA levels in each group were detected using real-time quantitative PCR.9 RAW264.7 cells were transfected with design plasmid to make it highly expressed p65,and then stimulate the cells with BCG,p19 m RNA levels in each group were detected by real-time quantitative PCR.Result:1 C57BL/6 mice were given BCG,and cell composition of lymph nodes was detected by flow cytometry.It was found that BCG led to the production of IFN-gamma CD4+T cells,the number of CD4+T IL-17 cells,the number of IFN-CD8+T cells were increased.2 The IL-23,IL-10,IL-1beta,IL-6 and IL-12 secretion were significantly increased in BMDM after stimulated by BCG.3 The secretion of IL-23 were also increased in mouse macrophage cell line RAW264.7 after stimulated by BCG,so that the expression of IL-23p19 m BNA were significantly increased.4 We found BCG at the transcriptional level promotes the expression of IL-23p19 and reduced the decay rate of IL-23p19 m RNA.In this experiments,primers containing IL-23p19 gene exon and intron were designed,real-time quantitative PCR method were used.5 The phosphorylated p65 level were significantly improved afetr BCG stimulated RAW264.7 cells by Western blotting,indicating that the NF-kappa B pathway may plays an important role in tuberculosis infection.6 The expression of m RNAp19 decreased significantly after using different levels of NF-kappa B pathway blocking agent SN50.The expression of m RNAp19 were significantly increased after transfected RAW264.7 cells with NF-kappa B p65 plasmid.Conclusion:1 BCG can lead to the increase of IFN-gamma CD4+T in mice,and can improve the production of IFN-gamma CD8+T cells and Th17 cells.2 BCG stimulation can make the increased secretion of IL-12,IL-6,IL-10,IL-23 and IL-1beta in BMDM.3 BCG can not only promote the transcription of IL-23p19 gene transcription,but also inhibit the decay process of the gene.4 BCG induced phosphorylation of NF-kappa B p65,and the signal pathway of NF kappa B played an important role in BCG induced m IL-23p19.