| Objective To study the effection of TLR2 in mycobacteria-induced macrophage apoptosis, We establish the model of macrophage infection with Mycobacterium in vitro, and observe the apoptosis of macrophage which induced by Mycobacteriums infection. The amount of TLR2 in macrophage is detected by flow cytometry and Western blot.Methods U937 cells were cultured in vitro, and then phorbol myristate acetate (PMA) was used to induce U937 cells to phagocytosis. Functional U937 cells were infected by Mycobacterium intracellulare (M.in), Mycobacterium tuberculosis (MTB) and Mycobacterium abscessus (M.ab) by Mycobacterium tuberculosis bacteria. U937 cells in each group were extracted at Oh,4h,8h,24h,48h,72h time point. The sample was divided into two parts. The apoptosis rate of U937 cells are detected by flow cytometry. And the level of TLR2 was detected by flow cytometry and Western blot. TLR2 in U937 cells were blocked by Anti-TLR2 monoclonal antibody. We used different mycobacterium affect the U937 cell. Then the apoptosis rate of U937 cells of which TLR2 were blocked was detected by flow cytometry.Results (1) The apoptosis of U937 cells which infected by different mycobacteriums increased as time went by. The apoptosis rate of MTB group was 6.00±0.3, M.in group 6.36±0.25 and M.ab 6.63±0.44 at 4h time point. There was no significant difference between the apoptosis rate of these three mycobacterias and normal control group (P>0.05). At 8h time point, the difference between different mycobacterium became to be obviously. The apoptosis rate of M.in group was 10.47±0.65, M.ab group was 13.40±1.15. They were obviously higher than normal control group 7.18±0.7 (P<0.01). But the apoptosis rate of MTB group was 7.80±0.53, there was no significant difference compared with normal control group (P>0.05). At 72h time point, the difference between each mycobacterium became more significant. The apoptosis rate of M.in group was 18.64±1.41, M.ab group 37.70±1.31, which was significant higher than normal control 11.22±1.07 (P<0.01). But there was no significant between MTB group and normal control group. At 72h time point, the apoptosis rate of U937 of M.ab group is the highest, and M.in group is the second, MTB group was lower than others. (2) TLR2 of U937 cell membrane was low express at the beginning of mycobacterium infection. The fluorescence of MTB group, M.in group, M.ab group were 3.25±0.54,2.82±0.27,3.19±0.37, which was no significant difference compare with normal control group (P>0.05). TLR2 expression of U937 cells enhanced quickly in the first 4h after infect by mycobacteria. TLR2 expression of MTB group, M.in group, and M.ab group were 3.25±0.54,2.82±0.273.19±0.37 at 4h. There was no significant difference compared with normal control group (P>0.05). After the first 8h, the TLR2 expression of each mycobacterium became stable with slight fluctuations. At 72h, the TLR2 expression of each mycobacterium were different from each other, the order of TLR2 expression level in the arrangement of high to low was MTB, M.in, M.ab. The same results were detected by Western blot. (3)The TLR2 of U937 cells were blocked by anti-TLR2 monoclonal antibody. Then the cells were infected by different mycobacteriums. The apoptosis of U937 cells were measured by flow cytometry. At 72h, the apoptosis of U937 cells in MTB group, M.in group, M.ab group and normal control group were 12.37±0.57,13.67±0.56,17.39±0.98 and 11.21±0.66. Compared with unbloked U937 cells, the apoptosis rate of the blocked U937 cells infected by M.in and M.ab decreased significantly (P<0.05), while the apoptosis rate of the blocked U937 cells infected by MTB and normal control have no significant difference (P>0.05).Conclusion 1. Mycobacterium can induce the apoptosis of macrophage. The apoptosis rate of macrophage gradually increased. Different species of mycobacterium have different ability to induce macrophage apoptosis.2. The TLR2 of macrophage increased quickly in the first 4h after infecting by Mycobacterium. The macrophage infected by different Mycobacterium expressed different level of TLR2.3. TLR2 was not the direct reason for apoptosis of macrophage, but TLR2 took part in the initial activation of apoptosis. |
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