羊种布鲁杆菌诱导小鼠巨噬细胞的凋亡及caspase3、8、9对细胞凋亡的调控

Analysis of macrophage apoptosis induced by Brucella melitensis and the effects of caspases 3, 8 and 9

目的观察羊种布鲁杆菌强毒株16M和减毒株M5-90诱导小鼠巨噬细胞RAW264．7凋亡的差异性，以及半胱氨酸蛋白酶（caspase）3、8、9对细胞凋亡的调控作用。方法通过羊种布鲁杆菌16M、M5-90对小鼠巨噬细胞多重感染（细菌数：细胞数=100：1、10：1、50：1）的方式，找出最佳感染复数（MOI）。按最佳MOI=50：1建立布鲁杆菌16M、M5-90感染巨噬细胞的模型，分别在感染后2、4、8、12、24、48h后收集被感染的细胞，计数细胞内细菌菌落形成数量单位，流式细胞仪检测细胞凋亡率及caspase3、8、9对细胞凋亡的影响。结果在感染后2、4、8、12、24、48h，16M在细胞内细菌菌落形成数量分别为10^5.4、10^4.8、10^5.8、10^6.5、10^8.0、10^9.0，M5-90在细胞内细菌菌落形成数量分别为10^6.1、10^6.2、10^6.4、10^6.3、10^6.1、10^5.0，其中在感染后24、48h，强毒株16M较弱毒株M5-90在细胞内寄生菌菌落形成数量明显增加。在感染后2、4、8、12、24、48h，16M诱导的巨噬细胞凋亡率分别为（2．67±0．09）％、（13．13±0．3）％、（6．56±0．42）％、（6．49±0．28）％、（16．07±0．86）％、（24．23±1．67）％，M5-90诱导的巨噬细胞凋亡率分别为（3．62±0．02）％、（32．01±2．59）％、（17．58±0．44）％、（16．09±0．10）％、（62．53±2．70）％、（85．53±0．15）％。对照组巨噬细胞凋亡率分别为[（1.90±0．20）％、（1．92±0．16）％、（1．99±0．03）％、（2．48±0．11）％、（3．56±0．07）％、（5．26±0．33）％]，各组同时间两两比较差距均有统计学意义（P均〈0．01）。其中在感染后24-48h，弱毒株M5-90比强毒株16M更能促进巨噬细胞的凋亡。在感染后24h，对照组caspase3、8,9的表达量分别为（1．47±0．05）％、（1．52±0．02）％、（2．47±0．12）％，M5-90组caspase3、8、9的表达量分别为（9．70±0．46）％、（6．08±0．56）％、（35．08±1．64）％，caspase3、8、9的表达量与对照组相比明显增高（P均〈0．01）。在加入easpase3、8、9抑制剂后24h，对照组细胞凋亡率为（66．72±1．28）％，M5-90组caspase3、8、9的细胞凋亡率分别为（22．62±0．55）％、（53．15±1．85）％、（29．18±0．23）％，easpase3、9的细胞凋亡率与对照组相比明显降低（P均〈0．01）。结论布鲁杆菌强毒株16M和减毒株M5-90均能加速巨噬细胞凋亡，M5-90作用大于16M。在M5-90作用下，easpase3、8、9对巨噬细胞凋亡均有调控作用。

Objective To determine the difference of macrophage RAW264.7 apoptosis induced by Brucella melitensis virulent strain 16M and attenuated strain M5-90 and elucidate the regulatory role of caspases 3, 8 and 9. Methods The best multiplicity of infection （MOI） was determined through kinetic analysis of Brucella melitensis strain 16M and M5-90 induced mouse macrophages apoptosis（bacterium ： cell = 100 ： 1, 50 ： 1, 10 ： 1 ）. The infection model was established using the best MOI = 50 ： 1. The numbers of in vivo bacteria by colony formation units were calculated after macrophages were infected for different times, including 2, 4, 8, 12, 24 and 48 h, and the infected cells were collected. The ratios of apoptosis were detected and the regulation of caspases 3,8 and 9 in apoptosis pathway was elucidated by flow cytometry. Results The numbers of 16M in vivo bacteria were 10^5.4, 10^4.8, 10^5.8, 10^6.5, 10^8.0 and 10^9.0, respectively and of M5-90 were 10^6.1, 10^6.2, 10^6.4, 10^6.3, 10^6.1 and 10^5.0, respectively. The number of in vivo bacteria of 16M was significantly increased than that of M5-90 after infected for 24 h to 48 h. The ratios of apoptosis induced by 16M after infected for 2,4,8,12,24 and 48 h was （2.67 ± 0.09）%, （13.13 ± 0.30）%, （6.56 ± 0.42）%, （6.49 ± 0.28）%, （16.07 ±0.86）% and （24.23 ± 1.67）%, respectively, and by M5-90 was （3.62 ± 0.02）%, （32.01 ± 2.59）%, （17.58 ± 0.44）%, （16.09 ± 0.10）%, （62.53 ± 2.70）% and （85.53 ± 0.15）%, respectively, and by control group was [（1.90 ± 0.20）%, （1.92 ± 0.16）%, （1.99 ± 0.03）%, （2.48 ± 0.11）%, （3.56 ± 0.07）%, （5.26 ：1： 0.33）%]. The differences were statistically between groups in same time. The Brucella melitensis vaccine strain M5-90 was more powerful than virulent strain 16M in respect of inducing maerophage apoptosis after infected for 24 to 48 h. Twenty-four hours after infection, the expression of caspases 3, 8 and 9 was （1.47 ± 0.05）%, （1.52 ± 0.02）% and （2.47 ± 0.12）%, respectively, in control group and the expression was （9.70 ± 0.46）%, （6.08 ± 0.56）% and （35.08± 1.64）%, respectively, after infected for 24 h induced by M5-90. The expression of caspases 3, 8 and 9 was significantly higher than that control group （P 〈 0.01 ）. Twenty-four hours after given easpases 3, 8 and 9 inhibitor, apoptosis rate in control group was （66.72 ± 1.28）%, in M5-90 group was （22.58 ± 0.55）%, .（53.15 ± 1.85）% and （29.18 ± 0.23）%, respectively, and compared with control group, apoptosis rate of caspases 3, 8 and 9 was significantly lower（P 〈 0.01）. Conclusions Apoptosis of macrophage can be induced by Brucella melitensis virulent vaccine strain 16M and attenuated strain M5-90. M5-90 is stronger than that of strain 16M. Caspases 3, 8 and 9 can regulate macrophage apoptosis after M5-90 infection.