巨噬细胞耐多药结核分枝杆菌感染模型功能特点的观察

Observation on the functional characteristics of multidrug-resistant Mycobacterium tuberculosis-infected macrophage model

目的观察巨噬细胞耐多药结核分枝杆菌(multidrug-resistant Mycobacterium tuberculosis,MDR-Mtb)感染模型的吞噬和杀菌功能特点,探讨其在吞噬杀菌过程中免疫应答和代谢功能的变化,为完善巨噬细胞在耐多药结核病(multidrug-resistant tuberculosis,MDR-TB)发生和发展中的作用机制提供依据。方法分别建立巨噬细胞MDR-Mtb和H37Rv株感染模型,采用菌落形成单位(colony-forming unit,CFU)计数、液相芯片技术和Cholesterol Assay试剂盒分别观察MDR-Mtb对巨噬细胞吞噬和杀菌功能、分泌细胞因子[Th1因子(IL-12/23 p40、IL-27和TNF-α)、Th2因子(IL-6和IL-10)]和胆固醇代谢的影响。采用SPSS25.0软件进行统计学分析,应用均值±标准差(Mean±SD)表示,比较采用t检验或F检验。以P<0.05为差异有统计学意义。结果(1)MDR-Mtb感染巨噬细胞后,胞内CFU计数逐渐增高,至感染后24 h达峰值;胞外CFU计数逐渐降低,至感染后24 h达低值。感染后48 h,胞内CFU计数较感染后24 h降低,胞外CFU计数则较感染后24 h增高(P<0.05),均与感染后4 h相接近(P>0.05)。感染后8~48 h,MDR-TB组胞内CFU计数均低于H37Rv组,而胞外CFU计数则高于H37Rv组(P<0.05)。(2)MDR-TB组巨噬细胞上清液IL-12/23 p40、IL-27、TNF-α、IL-6和IL-10表达量均高于空白对照组(P<0.05)。与感染后4 h相比较,仅感染后24 h、48 h的TNF-α和IL-6表达量增高(P<0.05)。与H37Rv组相比较,MDR-TB组感染后48 h的IL-12/23 p40和TNF-α及感染后24 h的IL-6表达量降低,而其感染后48 h的IL-27表达量则增高(P<0.05)。(3)MDR-TB组感染后24 h、48 h胆固醇的表达量较感染后4 h降低,且低于空白对照组(P<0.05),但与H37Rv组各时间点胆固醇表达量相接近(P>0.05)。(4)当感染后24 h MDR-TB组胞内CFU计数达峰值时,仅TNF-α表达量达峰值;当感染后48 h MDR-TB组胞内CFU计数降低时仅IL-6表达量达峰值。当感染后MDR-TB组胞内CFU计数呈现先增高后降低的趋势时,胆固醇表达量呈现降低趋势。结论MDR-Mtb感染巨噬细胞后可诱导其吞噬和杀菌功能,刺激相关Th1和Th2因子高表达,利用和消耗胆固醇。但此作用弱于H37Rv标准株。

Objective To observe the characteristics of the phagocytosis and bactericidal function of multidrug-resistant Mycobacterium tuberculosis(MDR-Mtb)-infected macrophage model,and the changes of the immune response and metabolic function in the process of phagocytosis and bactericidal function,aiming to provide reference for studying the role and mechanism of macrophages in the occurrence and development of multidrug-resistant tuberculosis(MDR-TB).Methods We established MDR-Mtb and H37Rv-infected macrophage models,and used the colony-forming unit(CFU),Magnetic Luminex®Assay and Cholesterol Assay kit to observe the effects on phagocytosis and bactericidal function,the secretion of Th1(IL-12/23 p40,IL-27 and TNF-α)and Th2 cytokines(IL-6 and IL-10)and cholesterol metabolism.The data were analyzed by SPSS25.0 software.The data were expressed as Mean±SD and analyzed by t test or F test.P<0.05 was considered statistically significant.Results(1)After MDR-Mtb-infected macrophages,the intracellular CFU gradually increased and reached the highest at 24 h,while the extracellular CFU gradually decreased and reached the lowest at 24 h.The intracellular CFU at 48 h was lower than that at 24 h,while the extracellular CFU was higher than that at 24 h(P<0.05).Both intracellular and extracellular CFU at 48 h were close to those at 4 h(P>0.05).The intracellular CFU was lower than the H37Rv group at 8-48 h,while the extracellular CFU was higher than the H37Rv group(P<0.05).(2)The level of IL-12/23 p40,IL-27,TNF-α,IL-6 and IL-10 of MDR-TB group were higher than those of blank group(P<0.05),but the level of TNF-αand IL-6 at 24 h and 48 h were higher than that at 4 h(P<0.05).IL-12/23 p40 and TNF-αat 48 h and IL-6 at 24 h were lower than those of the H37Rv group,while IL-27 at 48 h was higher than that of the H37Rv group(P<0.05).(3)The levels of cholesterol of MDR-TB group at 24 h and 48 h were lower than those of 4 h and blank group(P<0.05),but the level of cholesterol was similar to the H37Rv group at any time(P>0.05).(4)TNF-αreached the highest when the intracellular CFU reached the highest at 24 h,and IL-6 reached the highest when the intracellular CFU decreased at 48 h.With the decreasing of cholesterol expression,the intracellular CFU increased and then decreased.Conclusions MDR-Mtb could induce the phagocytosis and bactericidal function of macrophages,increase the expression of Th1 and Th2 cytokines and promote the utilization and consumption of cholesterol,but this function was weaker than that of H37Rv strain.