Mycobacterium Tuberculosis에 감염된 사람의 단구 유래 대식세포에서 신호전달 및 기능 = Signal Transduction and Function in Humane Monocyte Derived Macrophage Infected with Mycobacterium Tuberculosis
Mycobacterium tuberculosis provided by Tuberculosis Research Institute was used to induce macrophage by using interferon-γ(IFN-γ) and lipopolysaccharide to monocyte from human peripheral blood. Four groups consisted of macrophage phagocyted by alive M. tuberculosis. macrophage phagocyted with killed M. tuberculosis. macrophage phagocyted with microbead. and macrophage without phagocyted(Figure 1-A, 1-B). Prostaglandin products. H_(2)O_(2) products. and intracellular calcium ion were measured by radioimmunoassay method. Cytofluor^(2300) fluorometer method. and confocal microscopy.
The viability of M. tuberculosis phygocyted was measured by reverse transcriptase(RT)-PCR to investigate produced protein difference during the phagocytosis. Finally. two-dimensional gel electrophoresis method was used to identify produced intracellular protein difference.
The results were summarized as follows.
1. There was a statistically significant differences in the amount of produced Prostaglandin among four groups(P<0.01). The amounts of produced prostaglandin by macrophage phagocyted with microbead and killed M. tuberculosis were 1.5 and 3.0 times higher than that of control group(173.23±23.45pg/ml) respectively. The amount of produced prostaglandin by macrophage phagocyted with alive M. tuberculosis was significantly higher than that of control group specially(P<0.0l). The amount of thromboxan B_(2)(TXB_(2)) and 6-keto-F1α by macrophage phagocyted with alive M. Tuberculosis were 1.9 and 2.7 times higher than that of control group respectively. However. the increased amounts were not as high as the increased amounts of Prostaglandin E_(2)(PGE_(2))(Table 2).
2. Table 3 shows macrophages and H_(2)O_(2) products by macrophages. The amount of H_(2)O_(2) produced were 1.4 to 3.1 times higher than that of negative control group(1.023±l05 C.F.U). The amount of produced H_(2)O_(2) in positive control group and experimental groups after treated with Phobol myristate acetate (PMA) was 4.3 to 5.2 times higher than that of negative control group. The amount of produced H_(2)O_(2) treated with PGE_(2) was lower than that of negative control group and the amounts of produced H_(2)O_(2) after treated with IFN-γ and interleukin (IL-2) were higher than that of negative control group. The amount of H_(2)O_(2) by macrophage phagocyted with alive M. tuberculosis showed the highest value regardless of stimulation reagents. There were statistically significant differences in the amount of produced H_(2)O_(2) between the control group and the experimental group with alive M. tuberculosis treated with PGE₂and IFN-γ especially(P<0.01)(Table 3).
3. The effect of prostaglandin increased with M. tuberculosis infection on intercellular calcium ion release was investigated. The amount of release was highest in macrophage phagocyted with alive M. tuberculosis followed by macrophage phagocyted with killed M. tuberculosis and macrophage phagocyted with microbead (Figure 2).
4. The result of viability of M. tuberculosis by RT-PCR showed that M. tuberculosis was able to survive three days (Figure 3-B). The result also showed that it was possible to identify the DNA upto six days after the culture regardless of M. tuberculosis survival(Figure 3-A).
5. The produced protein differences among the experimental groups was investigated using M tuberculosis infection in the second day of the viability decided by RT-PCR. The result showed that there was a protein found in groups phagocyted with killed and alive M. tuberculosis which was not found in microbead. The test result also indicated that there was a produced protein differences among the experimental groups and there was a protein that was specifically increased in alive M. tuberculosis groups(Figure 4-A.B.C).
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