The Study Of Apoptosis Of Dendritic Cells Induced By M. Bovis With Different Virulence

Bovine tuberculosis is a chronic consumptive zoonosis casued by Mycoabcterium tuberculosis complex, and is fixed in B list by OIE. The disease caused about 3 billion dollars of direct economic loss all over the world. According to statistics, more than 10% of the human tuberculosis is caused by Mycobacterium bovis. Bovine tuberculosis exerts a tremendous influence on the development of animal husbandry and human health, and is also a worldwide public health problem.In view of the instability of the detection of bovine tuberculosis and low protection force of the vaccine, the study of pathogenic mechanism of Mycobacterium bovis is imperative. In-depth study of pathogenic mechanism of bovine tuberculosis could provide more theoretical basis and guidance for the new diagnostic methods of bovine TB and vaccines. Mycobacterium bovis is the intracellular parasitic bacteria, so the cellular immunity and tuberculosis are closely related. The dendritic cell is one of the host cells of Mycobacterium bovis. At present, dendritic cells are the most powerful antigen presenting cells, and is the only antigen-presenting cell that can activate the initial T-lymphocyte in the body. The application of dendritic cells as a model cell of Mycobacterium bovis cells is of great significance for the study of pathogenic mechanisams of the bovis tuberculosis.Apoptosis or programmed cell death is the process of active cell death controlled by genes. Through apoptosis, the body can clear damaged cells or cells infected by pathogenic microorganisms to maintain a relatively stable internal environment in the body. The result of gene chip in our laboratory showed that Mycobacterium bovis with different toxicity caused varying degrees of apoptosis in dendritic cell, and virulent strain can induce a much lower percentage of apoptosis than BCG. We use flow cytometric analysis, the analysis of DNA fragmentation detected by ELSA, Real time RT-PCR, Wwstern blot, Evaluation of nuclear morphology by fluorescence microscopy to detect the apoptosis of dendritic cell infected by the Mycobacterium bovis.Therefore, this research started from the interaction of dendritic cells with Mycobacterium bovis, The main results were showed as follows:1. The detection of cell cycle of dendritic cellsThe DNA content of dendritic cells was determined by Flow cytometry following PI staining. Compared to the dendritic cells infected by M. bovis, a much higher sub G0/G1 peak of dendritic cells infected by BCG was observed. That is to say, the M. bovis inhibited the apoptosis of dendritic cells. Statistic analysis showed that the ratio of apoptotic cells infected by BCG was about 2.5 fold as high as the DCs infected by Mycobacterium bovis. Differences were considered significant at p<0.001.2.The testing of DNA fragmentation of dendritic cellsApoptosis of dendritic cells was assessed by determining the amount of DNA fragmentation by the ELISA. ELISA results showed that Infection with virulent M. bovis consistently resulted in less DNA fragmentation of dendritic cells than infection with Mycobacteria bovis attenuated strains BCG. Statistic analysis showed that the ratio of apoptotic cells infected by BCG was about 1.55 fold as high as the DCs infected by Mycobacterium bovis, Differences were considered significant at p<0.01. In a word, BCG can induce more apoptosis than Mycobacterium bovis.3. The detection of apoptotic genes by Real time RT-PCRFor the Real time RT-PCR, compared to the dendritic cell infected by virulent strain, the gene (caspase3, caspase8, caspase9, and Dffa) expression of dendritic cell infected by BCG showed up-regulated expression. In addition, the expression of the caspase3 and caspase9 increased about 2.5 fold, and the gene expression of casapse8 and Dffa is about 1.2-1.3 fold. That is to say, virulent strain inhibited the apoptosis of dendritic cells. In addition, the caspase9 of dendritic cells infected by BCG showed a much higher up-rugulated expression, and the caspase9 is the critical apoptotic gene involving in the intrinsic apoptosis pathway. So the intrinsic apoptosis pathway may participate in the apoptosis of dendritic cells infected by BCG. The confirmative experiments are in progress.4. The detection of apoptotic genes by Western blotFor the Western blot, compared to the control group, the gene (caspase8, caspase9) expression of dendritic cell infected by BCG and virulent strai showed up-regulated expression. Compared to the dendritic cell infected by virulent strain, the gene (caspase8, caspase9) expression of dendritic cell infected by BCG showed up-regulated expression. That is to say, both BCG and virulent strain can induce apoptosis of dendritic cells, and compared to the BCG group, virulent strain inhibited the apoptosis of dendritic cells. In addition, The gene caspase9 of dendritic cells infected by BCG showed a much higher expression in protein level. So the intrinsic apoptosis pathway may participate in the apoptosis of dendritic cells infected by Mycobaterium bovis.5. Evaluation of nuclear morphology by fluorescence microscopy Fluorescence microscopy of Hoechst33342-stained infected cells indicated that cell shrinkage and nuclear condensation, which is the typical features of apoptosis occurred.6. The expression of cytokine of dendritic cells infected by Mycobaterium bovisThe level changes of some cytokines were also tested with ELISA kits, including IL-1, IL-10, IL-12, TNF-α.etc. The dendritic cells infected by BCG and M. bovis secreted similar amout of IL-10.There is no significant difference(P>0.05). The dendrtic cells infected by BCG secreted 1.6 fold more TNF-a than those of Virulent M. bovis group, Significant difference(P<0.0.1). Virulent M. bovis infected dendritic cells secreted 2 fold (P<0.01) and 1.3 fold (P<0.05)more IL-1 and IL-12 than those ofBCG infected dendritic cells.