TLR2和TLR6在鸟分枝杆菌诱导巨噬细胞凋亡中的作用

摘要

[目的]建立鸟分枝杆菌感染巨噬细胞模型,探讨 Toll 样受体 2(TLR2)和 Toll 样受体 6(TLR6)在鸟分枝杆菌诱导巨噬细胞凋亡中的作用,为阐明鸟分枝杆菌致病机制提供依据.[方法]用鸟分枝杆菌感染 U9 3 7 细胞,于感染0 h、8 h、16 h、24 h和48 h后分别提取细胞总蛋白,并通过 Western blot方法检测TLR2 和TLR6 的表达;分别提取细胞培养上清液,利用 ELISA技术检测肿瘤坏死因子(TNF-α)含量的变化情况.用鸟分枝杆菌感染阻断TLR2 和TLR6 后的 U937 细胞,通过 Western blot 方法分别检测其促凋亡因子 BAX 和抗凋亡因子BCL-2 的表达量,用 ELISA技术检测细胞培养上清液中TNF-α含量变化情况,用流式细胞仪检测 U9 3 7 细胞的凋亡变化.[结果]鸟分枝杆菌感染U9 3 7 细胞可引起 TLR2 、TLR6 和TNF-α表达上调;用鸟分枝杆菌感染阻断TLR2 和TLR6 后的 U937 细胞,可使 U937 细胞内促凋亡因子BAX和TNF-α表达量下降(P＜0.05),抗凋亡因子BCL-2 表达量升高(P＜0.05),并可以明显降低感染鸟分枝杆菌后的细胞的凋亡率(P＜0.05).[结论]TLR2 和TLR6 与巨噬细胞凋亡相关,巨噬细胞通过 TLR2 和 TLR6 来识别鸟分枝杆菌,而鸟分枝杆菌反过来通过促进TLR2 和TLR6 的表达来影响巨噬细胞相关凋亡通路,从而诱导巨噬细胞凋亡.%[Objective]A Mycobacterium avium-infected macrophage model was established to investigate the role of Toll-like receptor 2(TLR2)and Toll-like receptor 6(TLR6)in the ap-optosis of macrophages induced by Mycobacterium avium and to provide a basis for clarifying the pathogenesis of Mycobacterium avium.[Methods]U937 cells were infected with Mycobacterium avium at 0 h,8 h,16 h,24 h,and 48 h time points,respectively,and then the total proteins of the cells were used to detect the ex-pression of TLR2 and TLR6 by Western blot.The cell culture supernatants were extracted re-spectively and the changes of tumor necrosis fac-tor(TNF-α)content were detected by ELISA.The cytokines TNF-αwas measured by ELISA from U937 cells supernatant.TLR2 and TLR6 monoclonal antibody were used to treat the U937 cells 1 h before Mycobacterium avium stimu-lation.The expression of BAX and BCL-2 was evaluated by Western blot.The cytokines of TNF-αwas measured by ELISA from U937 cells supernatant.U937 cell apoptosis rate was de-tected with flow cytometry.[Results]Mycobacterium avium infection of U937 cells could cause the upregulation of TLR2,TLR6,and TNF-α expression.Blocking U937 cells after TLR2 and TLR6 infection with Mycobacterium avium could cause the expression of proapoptotic factors BAX and TNF-αin U937 cells.The amount was decreased(P＜0.05),the expression level of anti-apoptosis factor BCL-2 was increased(P＜ 0.05),and the apoptosis rate of cells infected with Mycobacterium avium was significantly decreased(P ＜ 0.05).[Conclusion]TLR2 and TLR6 are associated with macrophage apoptosis,macrophages recognize M.avium by TLR2 and TLR6,whereas Mycobacterium avium in turn affects macrophage-associated apoptosis path-ways by promoting the expression of TLR2 and TLR6.