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目的探讨结核分枝杆菌MPT64抗原对RAW264.7巨噬细胞的影响及相关机制。方法将MPT64基因在大肠杆菌中表达、纯化,获得的纯化蛋白用于后续实验。将用佛波醇酯(PMA)分化的RAW264.7巨噬细胞分为对照组、卡介菌纯蛋白衍生物(BCGPPD)诱导组、BCG-PPD+MPT64共同作用组,分别给予相应的处理。孵育16 h后,采用流式细胞术检测细胞凋亡情况,用ELISA试剂盒检测培养细胞上清的细胞因子TNF-α及IL-10水平。结果 BCG-PPD可以诱导RAW264.7巨噬细胞凋亡,BCG-PPD+MPT64组的细胞凋亡水平低于BCG-PPD组(P<0.05)。细胞因子上清检测结果表明,与对照组相比,BCG-PPD作用组的TNF-α水平升高(P<0.01),而IL-10水平无明显变化;与BCG-PPD组相比,BCG-PPD+MPT64共同孵育组的IL-10水平升高(P<0.01),而TNF-α水平无明显变化。结论 MPT64抗原可能是一种毒力因子,能够抑制BCG-PPD诱导的巨噬细胞凋亡,该作用可能是通过提高IL-10水平来实现的。
Objective To investigate the effect of Mycobacterium tuberculosis MPT64 antigen on RAW264.7 macrophages and related mechanisms. Methods The gene of MPT64 was expressed in Escherichia coli and purified. The purified protein was used in subsequent experiments. RAW264.7 macrophages differentiated by phorbol ester (PMA) were divided into control group, BCGPD induced group and BCG-PPD + MPT64 combined group, respectively. After 16 h of incubation, the apoptosis of cells was detected by flow cytometry. The levels of cytokines TNF-α and IL-10 in the supernatant of cultured cells were detected by ELISA kit. Results The apoptosis of RAW264.7 macrophages was induced by BCG-PPD. The apoptosis of BCG-PPD + MPT64 group was lower than that of BCG-PPD group (P <0.05). The result of cytokine supernatant showed that compared with the control group, the level of TNF-α in BCG-PPD group increased (P <0.01), while the level of IL-10 did not change significantly. Compared with BCG-PPD group, -PPD + MPT64 co-incubated group IL-10 levels increased (P <0.01), while TNF-α levels did not change significantly. Conclusion MPT64 antigen may be a virulence factor that can inhibit the apoptosis of macrophages induced by BCG-PPD, which may be achieved by increasing the level of IL-10.