结核分枝杆菌MPT64抗原对巨噬细胞的影响及其与Toll样受体-2的相关性

Effect of MPT64 from Mycobacterium Tuberculosis on Macrophage and Expression of Toll-like Receptor 2

目的观察结核分枝杆菌MPT64抗原对RAW264.7巨噬细胞凋亡及Toll样受体-2(toll-like receptor 2,TLR-2)表达的影响。方法将佛波肉荳蔻醋酸(phorbol myristoyl acetate,PMA)分化的RAW264.7巨噬细胞分为阴性对照组、卡介菌纯蛋白衍化物(purified protein derivative of BCG,BCG-PPD)诱导组、PPD+MPT64共同作用组,孵育16 h后,Annexin V-FITC染色,用流式细胞技术(flow cytometry,FCM)和Western blot检测细胞凋亡、细胞膜及细胞内TLR-2的表达情况。结果 BCG-PPD可以诱导RAW264.7巨噬细胞凋亡,PPD+MPT64作用组的细胞凋亡水平明显低于BCG-PPD组,随着MPT64蛋白浓度的逐渐增高,凋亡水平逐渐降低。FCM和Western blot技术检测结果表明,BCG-PPD组的TLR-2表达水平明显高于阴性对照组,在诱导16 h达到高峰。而在PPD+MPT64组,细胞膜及细胞内的TLR-2水平均明显低于BCG-PPD组。结论 MPT64抗原能够抑制PPD诱导的巨噬细胞凋亡,可能通过调节TLR-2的表达水平来实现。MPT64抗原可能是一种毒力因子,在结核分枝杆菌感染机体的过程中,通过抑制巨噬细胞凋亡参与宿主与结核分枝杆菌的相互作用。

Objective To explore the effect of mycobacterial antigen MPT64 on the apoptosis of RAW264. 7 macrophage and toll-like receptor 2 （TLR-2） expression. Methods The RAW264. 7 differentiated by phorbol myristoyl acetate （PMA） were divided into negative,PPD group, BCG PPD induced group and combined PPD and MPT64 group. After incubation for 16 hours, the cell apoptosis and TLR-2 expression were detected by flow cytometry after dying by Annexin V-FITC and Western blot. Results BCG-PPD could induce the apoptosis of the RAW264. 7 cells. The apoptosis level in combined PPD and MPT64 group was significantly lower than that in PPD group and decreased along with the increase of MPT64 protein concentration. TLR-2 expression levels was higher in PPD group than that in the negative group and arrived at the maximum at 16 hours after induction. TLR-2 expression in the cell membrane and cells decreased in PPD ＋ MPT group was significantly lower than that in PPD group. Conclusion MPT64 can inhibit the apoptosis induced by PPD, which might be realized by modulating TLR-2 expression. MPT64 might be a virulence factor, and participate in the interaction between the host and Mycobacterium tuberculosis by inhibition of macrophage apoptosis.