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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 observe the effect of Mycobacterium tuberculosis MPT64 antigen on the apoptosis of RAW264.7 macrophages and the expression of toll-like receptor 2 (TLR-2). Methods The RAW264.7 macrophages differentiated from phorbol myristoyl acetate (PMA) were divided into negative control group, BCG-PPD induced group, PPD + MPT64 group. After 16 h of incubation, Annexin V-FITC staining was used to detect the expression of TLR-2 in the cell membrane and the cell membrane by flow cytometry (FCM) and Western blot. Results The apoptosis of RAW264.7 macrophages was induced by BCG-PPD. The apoptosis of cells treated with PPD + MPT64 was significantly lower than that of BCG-PPD. With the increase of MPT64 protein concentration, the apoptotic rate decreased gradually. The results of FCM and Western blot showed that the expression level of TLR-2 in BCG-PPD group was significantly higher than that in negative control group, reaching the peak at 16 h after induction. In PPD + MPT64 group, the levels of TLR-2 in cell membrane and cell were significantly lower than those in BCG-PPD group. Conclusion MPT64 antigen can inhibit PPD-induced apoptosis of macrophages, which may be through the regulation of TLR-2 expression level. The MPT64 antigen may be a virulence factor involved in the interaction of host with M. tuberculosis by inhibiting macrophage apoptosis in the course of M. tuberculosis infection.