结核分枝杆菌抗原激活核因子κB延迟中性粒细胞自发凋亡的作用

Mycobacterium tuberculosis antigen activates the nuclear factor-κB pathway and delays spontaneous apoptosis in neutrophils

目的探讨结核分枝杆菌抗原(Mtb-Ag)对中性粒细胞自发凋亡的影响。方法取健康成人外周血分离中性粒细胞,加 Mtb-Ag 培养24 h,或用核因子κB(NF-κB)抑制剂 N-甲苯磺基-L-苯乙胺酰氯甲基酮(TPCK)预处理30 min 后,膜联蛋白 V(AnnexinV/PI)染色,流式细胞术观察细胞凋亡情况;并用凝胶电泳迁移率变动分析(EMSA)化学发光法检测 Mtb-Ag 刺激0、1、2、4、6、24 h 后,中性粒细胞 NF-κB的 DNA 结合活性。结果加 Mtb-Ag(1.125 mg/ml)培养的中性粒细胞凋亡率(32%±3%)与中性粒细胞体外培养24 h 后自发凋亡率(55%±6%)相比,明显降低(P<0.05)。Mtb-Ag(1.125 mg/ml)作用中性粒细胞1 h 后明显可见 NF-κB活化,2 h NF-κB的 DNA 结合活性达高峰,24 h 回到静息水平;经 TPCK(30μmol/L)预处理后可阻断 Mtb-Ag 的抑制凋亡作用。结论经激活 NF-κB介导后,Mtb-Ag 对中性粒细胞自发凋亡有抑制作用。

Objective To explore the effect of Mycobacterium tuberculosis antigen （Mtb-Ag） on neutrophils apoptosis. Methods The fresh isolated neutrophils from healthy adults blood were cultured with Mtb-Ag for 24 h, with or without pretreatment of nuclear factor -κB （NF-κB） inhibitor N-tosyl-L-phenylanyl chloremethyl ketone （TPCK） for 30 minutes. Annexin V staining and Flow cytometry were used to measure cell apoptosis of neutrophils. NF-κB DNA binding was measured by gelelectrophorestic mobility shift assay （EMSA） in neutrophils after incubated with Mtb-Ag for 0,1,2,4,6,24 hours. Results Comparing to the spontaneous apoptosis （55%± 6% ） of neutrophils after culture in vitro for 24 h, treatment of Mtb-Ag （1. 125 mg/ml） decreased the cell apoptosis of neutrophils （32%±3% ）. The NF-κB shift bands were detected at 1 h in neutrophils after stimulated by Mtb-Ag, and reached maximum peak at 2 hours, and then returned to basal levels within 24 h. Pretreatment of TPCK inhibited the anti-apoptosis role of Mtb-Ag in neutroph/ls. Conclusion Mtb-Ag prevents neutrophils apoptosis and its inhibitory role concerns NF-κB pathway.