摘要
目的探讨结核分枝杆菌（MTB）小分子热休克蛋白Hspl6．3表达与感染小鼠肺泡巨噬细胞凋亡的关系。方法分别将MTB国际标准强毒株H37Rv株Hspl6．3基因缺失突变菌株（△H37Rv）、结核杆菌国际标准强毒株H37Rv菌株（H37Rv）以及无菌生理盐水溶液（空白对照）通过小鼠尾静脉注入小鼠体内以建立小鼠的感染模型，并在感染后的1、3、5、7、9、11、13、15d分离及鉴定小鼠肺泡巨噬细胞，用激光共聚焦显微镜技术检测及鉴定MTB感染小鼠肺泡巨噬细胞；流式细胞术检测不同时间点感染小鼠肺泡巨噬细胞的凋亡率；Westernblot技术检测小鼠肺泡巨噬细胞内半胱天冬酶-3（Caspase-3）和B细胞淋巴瘤／白血病-2（Bel-2）蛋白的表达。结果△H37Rv菌株感染组和H37Rv菌株感染组凋亡率均高于空白对照组，△H37Rv组的巨噬细胞凋亡率在感染1-7d内逐渐升高，至感染7d时达到高峰，随后呈下降趋势，与H37Rv组相比，1-7d及13-15d，△H37Rv组的巨噬细胞凋亡率高于H37Rv组，差异有统计学意义（P〈0．05）；H37Rv组和△H37Rv组的巨噬细胞Caspase-3和Bcl2蛋白表达水平均高于空白对照组；l-7d内，H37Rv感染组和△H37Rv感染组的巨噬细胞Caspase-3表达水平逐渐升高，至第7天达到高峰，且△H37Rv感染组在第13天时出现第二次高峰，但Caspase-3表达水平均高于H37Rv感染组，差异有统计学意义（P〈0．05）；在感染的早期（1-7d），△H37Rv感染组Bcl-2的表达水平无明显变化（P〈o．05），但在9d后逐渐升高；H37Rv感染组巨噬细胞内Bcl-2的表达水平1-7d内无明显变化（P〈O．05），7d后逐渐升高，但△H37Rv感染组Bcl-2的表达水平始终低于H37Rv组且7d后表现更为显著。结论在MTB感染的早期和晚期，MTB小分子热休克蛋白Hspl6．3的表达能够有效抑制小鼠肺泡巨噬细胞的凋亡，而这种抑制作用可能是通过抑制凋亡蛋白酶Caspase-3的表达，同时促进Bcl-2蛋白的表达来实现的。
Objective To study the relationship between the expression of Mycobacterium tuberculosis small heat shock protein Hsp16.3 and the apoptosis of infected mouse alveolar macrophages. Methods The laboratory mice were infected with bacterial suspension of the international standard virulent strain of Mycobacterium tuberculosis H37Rv strains （H37Rv）, Hsp16. 3 gene deletion mutants of the international standard virulent of Mycobacterium tuberculosis H37Rv strains（△H37Rv）, or sterile saline solution （normal control） by the tail vein. After successful replication of mouse infection model in each group, we cleaved the alveolus of each group of mice andcollected lavage fluid to obtain alveolar macrophages of the infected mice at days 1, 3, 5, 7, 9, 11, 13 and 15. Then the infection status of macrophages was observed with confocal laser scanning microscopy; flow cytometry was used to detect the apoptosis rate of alveolar macrophages of the infected mice; Caspase-3 and Bcl-2 expressions were examined by Western blot. Results The apoptosis rate of Hsp16.3 gene was higher in deletion strain （△H37Rv） group and H37Rv strains （H37Rv） group than in control group. The apoptosis rate of alveolar macrophages in △ H37Rv group gradually increased, peaked at day 7, and then gradually decreased. It was significantly higher in H37Rv group than in H37Rv strain group from day 1 to 7 and from day 13 to 15 （P〈0.05）. Caspase-3 and Bcl-2 protein expressions in the macrophages of AH37Rv group and H37Rv group were higher than those of control group. Caspase-3 expression in the microphages of △H37Rv group and H37Rv group gradually increased from day 1 to 7 and peaked at day 7; it peaked again at day 13 in H37Rv group. However, Caspase-3 expression remained significantly higher in△H37Rv group than in H37Rv group （P〈0. 05）. Bcl-2 expression in△H37Rv group did not change much at the early stage of infection （P〈0.05）, but gradually increased after day 9. Bcl-2 expression in H37Rv group did did not change much from day 1 to 7 （P〈0.05）, but gradually increased after day 7. However, it remained lower in/kH37Rv group than in H37Rv group, especially after 7 days（P〈0.05）. Conclusion Mycobacterium tuberculosis small heat shock protein Hsp16.3 can inhibit the apoptosis of macrophages during the early and late stages of infection, and this inhibition may be achieved by inhibiting the expression of apoptotic protease Caspase-3 and promoting the expression of Bcl-2 orotein.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2014年第3期300-305,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.81260261
81160192
30960355)
新疆生产建设兵团医药专项资金资助项目(No.2012BA022)
石河子大学科学技术研究发展计划"自然科学与计划创新"重点项目(No.ZRKX2010ZD01)~~
