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探讨结核分枝杆菌小分子热休克蛋白Hsp16.3基因缺失突变菌株对感染小鼠肺泡巨噬细胞凋亡率的影响及其时相性变化。用激光共聚焦显微镜技术检测及鉴定结核分枝杆菌感染小鼠肺泡巨噬细胞,流式细胞术检测不同时期感染小鼠肺泡巨噬细胞凋亡率,比较结核分枝杆菌Hsp16.3基因缺失突变株与正常株感染小鼠肺泡巨噬细胞凋亡率的时相性变化。激光共聚焦显微镜检测结果显示:结核分枝杆菌国际标准强毒株H37Rv株、结核分枝杆菌国际标准强毒株H37Rv株Hsp16.3基因缺失突变株(△H37Rv)、卡介苗菌株(BCG)以及卡介苗株Hsp16.3基因缺失突变株(△BCG)均被小鼠肺泡巨噬细胞大量吞噬。流式细胞技术检测结果显示:小鼠感染△H37Rv菌株后,巨噬细胞的凋亡率逐渐上升,至感染7d时达到高峰,随后逐渐降低,1～7d内,各时间段△H37Rv感染组巨噬细胞凋亡率均显著高于H37Rv感染组,9～11d内,△H37Rv感染组巨噬细胞凋亡率反而低于H37Rv菌株组,但13～15d内,△H37Rv感染组巨噬细胞凋亡率又呈现出比H37Rv感染组高的现象,差异均有统计学意义(P<0.05)。△BCG组凋亡率1～7d内呈现明显下降趋势,7d后巨噬细胞凋亡率变化趋于平稳,且1～5d内,△BCG组凋亡率显著高于BCG组(P<0.05),7～15d内,△BCG组与BCG组巨噬细胞凋亡率无明显差异。结果表明:与结核分枝杆菌H37Rv株野生株相比,结核分枝杆菌H37Rv株Hsp16.3基因缺失突变株在感染的早期和晚期对小鼠肺泡巨噬细胞有更强的致凋亡作用,而这种致凋亡作用与结核分枝杆菌小分子热休克蛋白Hsp16.3基因的缺失相关,说明在结核分枝杆菌感染的早期和晚期,结核分枝杆菌小分子热休克蛋白Hsp16.3的表达能够抑制宿主巨噬细胞的凋亡。
To investigate the effect of Mycobacterium tuberculosis small molecule heat shock protein Hsp16.3 deletion mutant on the apoptosis rate of alveolar macrophages in infected mice and the change of its phase. Laser confocal microscopy was used to detect and identify the alveolar macrophages in mice infected with Mycobacterium tuberculosis. The apoptosis rate of alveolar macrophages in mice infected by Mycobacterium tuberculosis was detected by flow cytometry. The deletion mutation of Mycobacterium tuberculosis Hsp16.3 gene was compared Time - course changes of apoptosis rate of alveolar macrophages in mice infected with strains and normal strains. Confocal laser scanning microscopy showed that the H37Rv strain of Mycobacterium tuberculosis, H37Rv strain of H37Rv strain of Mycobacterium tuberculosis (H37Rv), BCG (BCG) and BCG Strain Hsp16.3 gene deletion mutant (△ BCG) were phagocytosed by mouse alveolar macrophages. The results of flow cytometry showed that the apoptotic rate of macrophages increased gradually after infected with △ H37Rv strain and reached a peak at 7 days after infection, and then gradually decreased. Within 1 ~ 7 days, The apoptosis rate of macrophages in H37Rv infection group was significantly higher than that in H37Rv infection group in 9 to 11 days. However, the apoptosis rate of macrophage in △ H37Rv infection group was lower than that in H37Rv infection group, but within 13 ~ 15 days, the apoptosis of macrophage in △ H37Rv infection group Rates again showed higher than the H37Rv infection group, the differences were statistically significant (P <0.05). The apoptosis rate of △ BCG group showed a significant decrease within 1-7 days, and the apoptosis rate of macrophage cells tended to be stable after 7 days. The apoptotic rate of △ BCG group was significantly higher than that of BCG group within 1 ~ 5 days (P <0.05) , 7 ~ 15d, △ BCG group and BCG group macrophage apoptosis rate was no significant difference. The results showed that compared with the wild type strain of Mycobacterium tuberculosis H37Rv strain, the Hsp16.3 gene deletion mutant of Mycobacterium tuberculosis H37Rv strain had a stronger apoptosis-inducing effect on mouse alveolar macrophages in the early and late stages of infection, This apoptosis-inducing effect is related to the deletion of the Hsp16.3 gene of Mycobacterium tuberculosis small heat shock protein, indicating that in the early and late stage of Mycobacterium tuberculosis infection, the Mycobacterium tuberculosis heat shock protein Hsp16.3 Expression can inhibit the apoptosis of host macrophages.