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

The Effect of Mycobacterium tuberculosisSmall Heat Shock Proteins Hsp16.3 Gene-deleted Mutant Strains on Infected Mice Alveolar Macrophage Apoptosis Rate and Its Temporal Changes

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 heat shock proteins Hsp16.3 gene-deleted mutant strains on infected mice alveolar macrophage apoptosis rate,and evaluate its temporal changes.Infectious status of macrophages was observed with con-focal laser scanning microscopy（CLSM）,flow cytometry was used to detect alveolar macrophages apoptosis rate of the infected mice.Results showed that the international standard virulent strain of Mycobacterium tuberculosis H37Rv strains（H37Rv）,Hsp16.3 gene-deleted mutants of the international standard virulent of Mycobacterium tuberculosis H37Rv strains（△H37Rv）,BCG strains（BCG） and Hsp16.3 gene-deleted mutant of the BCG strains（△BCG） were swallowed largely by alveolar macrophages of the infected mice,alveolar macrophage apoptosis rate of the mice infected with △H37Rv gradually increased and peaked in on the 7th day,then gradually decreased,alveolar macrophage apoptosis rate of △H37Rv strain group were significantly higher than H37Rv strain group from 1d to 7d（P0.05）.△H37Rv strain group is lower than the H37Rv strain group from 9d to 11d,but alveolar macrophage apoptosis rate of △H37Rv strain groups recovered much higher than the H37Rv strain group from 13d to 15d,the differences were statistically significant（P0.05）.The alveolar macrophage apoptosis rate of the △BCG group presented a clear downward trend from 1d to 7d,the macrophage apoptosis rate steadily changed after 7th day,the macrophage apoptosis rate of △BCG group significantly higher than the BCG group from 1d to 5d（P0.05）.The macrophage apoptosis rate of △BCG group and BCG group was not significantly different from 7d to 15d.These results suggest that compared with the wild-type strain of Mycobacterium tuberculosis H37Rv strain,H37Rv strain of Mycobacterium tuberculosis Hsp16.3 gene-deleted mutants has stronger apoptosis induced on alveolar macrophages of mice in the early and late stages of infection,which is related to deletion of Mycobacterium tuberculosis small heat shock protein Hsp16.3,and Mycobacterium tuberculosis small heat shock protein Hsp16.3 could inhibit apoptosis of macrophages during the early and late stages of infection.