Research For The Mechanism Of Mycobacterium Tuberculosis Induced Macrophage Apoptosis

Mycobacterium tuberculosis (Mtb) was a major health problem of the world in the past, untill eifective chemical therapy was invented. But with the increase of the world population, acceleration of populace migration and HIV infection, tuberculosis situation has deteriorated in the last decade. Nowadays, many studies on pathogenesis of Mtb infection and the interaction between macrophages and pathogens have being carried out. And the apoptosis of macrophages/monocytes of the infectious murine has emerged as an important event in the host-parasite relationship. In this research, the murines were infected with several pathogens including M. tuberculosis H37Rv, H37Ra and M. bovis BCG. The apoptosis of murine macrophage, cellular signal molecules of some pathway in M. tuberculosis and in other mycobacteria were examined.M. tuberculosis H37Rv, H37Ra and M. bovis BCG were grown at 37℃ in Lowenstein-Jenson culture for 3-5 weeks. Cultures were collected when a thin surface layer were formed. Bacterial clumps were disrupted in 0.9 percent saline solution(l mg/mL), and each mice was injected 0.3mL through caudal vein. The broncho- alveolar lavage were acquired at different time points. The fluid was centrifuged and the macrophages were then separated. The cells were respectively reserved in 70 percent ethanol solution, 2 percent glutara solution and 4 percent citromint solution. Morphological change of macrophages observed with transmission electron microscopy and fluorescence staining. Agarose gel electrophoresis was used as the qualitatively detection . The apoptosis rate, Toll-like receptor 2(TLR2) and Bcl-2 expression were detected by flow cytometry.The results showed that. In the virulent (H37Rv) group of 5 days after infection, many macrophages exhibited the characteristics of apoptosis such as cell volume becoming smaller, nuclear membrane expanding apparently and chromatin attaching to the nuclear membrane. In the fluorescence staining, the nuclear of the apoptotic cells became diminution, some nuclears broke into several fragments. There were manyapoptotic cells existed in group H37Rv, but some apoptotic cells could be found in group H37Ra and group BCG which could be attributed to the metabolism of the body. In the Agarose gel electrophoresis, the characteristic DNA " Ladder " only existed in virulent (H37Rv) group. It the flow cytometry, the apoptosis rate of virulent (H37Rv) group gradually increased from 1 to 7 days, with the apoptosis rate of 32.2% at 7 days after infection, and then declined after 9 days . Bcl-2 expression of group H37Rv was higher than that of BCG group after 11 days. The TLR2 expression of group H37Rv and group BCG were higher than that in control group, but there was no obvious changes of each group at different time pointsMycobacterium tuberculosis (Mtb) H37Rv strain could apparently induce apoptosis of macrophages at the early phage of infection, and the high expression of Bcl-2 protein could inhibit apoptosis. The level of TLR2 has no obvious changes indicating that the during the infection course, the apoptosis of host macrophage may not depend on the TLR2 signal pathway primarily. Some other signal pathway and agents may affect the apoptosis course of the host macrophages in vivo.