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目的:探讨Mcl-1信号通路阻断剂在结核分枝杆菌H37Rv感染小鼠模型中对Mcl-1表达、巨噬细胞凋亡情况及结核分枝杆菌的影响。方法:小鼠腹腔注射H37Rv菌悬液,建立感染小鼠模型,针对Mcl-1的信号通路选用JAK/STAT信号通路阻断剂AG490、MAPK信号通路阻断剂PD98059和PI3K信号通路阻断剂LY294002用腹腔注射方式作用于各组感染小鼠模型,分为H37Rv感染组、AG490处理组、PD98059处理组、LY294002处理组和对照组。通过细胞抗酸染色观察结核分枝杆菌H37Rv感染小鼠腹腔巨噬细胞的动物模型是否建立成功;通过免疫细胞化学检测结核分枝杆菌H37Rv感染巨噬细胞的Mcl-1表达情况,使用流式细胞技术检测各组巨噬细胞的凋亡率,采用结核分枝杆菌菌落计数来判断巨噬细胞凋亡对结核分枝杆菌的清除效果。结果:细胞抗酸染色结果可见感染的巨噬细胞内散在排列的红色短小抗酸结核分枝杆菌。免疫细胞化学结果显示H37Rv感染组、AG490处理组和LY294002处理组中的Mcl-1蛋白为强阳性表达,PD98059处理组中Mcl-1蛋白为弱阳性表达,对照组Mcl-1蛋白为阴性表达。流式细胞术检测发现H37Rv感染组巨噬细胞凋亡率较对照组高,PD98059处理组的凋亡率显著高于各组,差异显著(P<0.05)。结核分枝杆菌菌落计数结果显示PD98059处理组对H37Rv菌株抑菌作用最明显。结论:Mcl-1信号通路阻断剂通过抑制JAK/STAT、MAPK和PI3K信号通路增加结核分枝杆菌H37Rv感染巨噬细胞的凋亡率,抑制结核分枝杆菌生长;其中,MAPK信号通路干扰Mcl-1的作用最明显,感染的巨噬细胞凋亡率最高,抑菌作用最强。
Objective: To investigate the effects of Mcl-1 signaling pathway inhibitor on the expression of Mcl-1, apoptosis of macrophages and Mycobacterium tuberculosis in a mouse model of Mycobacterium tuberculosis H37Rv infection. Methods: Mice were injected intraperitoneally with H37Rv suspension to establish an infected mouse model. The signal pathways of Mcl-1 were JAK / STAT signaling pathway inhibitor AG490, MAPK signaling pathway inhibitor PD98059 and PI3K signaling pathway inhibitor LY294002 The model of mice infected by intraperitoneal injection was divided into H37Rv infection group, AG490 treatment group, PD98059 treatment group, LY294002 treatment group and control group. To observe whether M tuberculosis H37Rv infected peritoneal macrophages in mice was successfully established by cell antacid staining and Mcl-1 expression in macrophages infected with Mycobacterium tuberculosis H37Rv by immunocytochemistry. Flow cytometry Technology to detect the apoptosis rate of macrophages in each group, the use of Mycobacterium tuberculosis count to determine macrophage apoptosis Mycobacterium tuberculosis clearance effect. Results: The results of cell acid-fast staining showed that the scattered red short-chain Mycobacterium tuberculosis in infected macrophages. Immunocytochemistry showed that Mcl-1 protein in H37Rv infected group, AG490-treated group and LY294002-treated group was strongly positive, while Mcl-1 protein in PD98059-treated group was weakly positive and Mcl-1 protein was negative in control group. Flow cytometry showed that the apoptosis rate of macrophages in H37Rv infection group was higher than that in control group, and the apoptosis rate in PD98059 group was significantly higher than that in control group (P <0.05). Mycobacterium tuberculosis colony counting results showed that PD98059 treatment group had the most obvious antibacterial effect on H37Rv strain. Conclusion: Mcl-1 signaling pathway blockers can inhibit Mycobacterium tuberculosis growth by inhibiting the JAK / STAT, MAPK and PI3K signaling pathways and increasing the apoptotic rate of Mycobacterium tuberculosis H37Rv infected macrophages. Among them, MAPK signaling pathway interferes with Mcl -1 is the most obvious, the highest rate of apoptosis of infected macrophages, the strongest antibacterial effect.