活动性肺结核相关巨噬细胞M1/M2极化的改变及ESAT6对巨噬细胞极化的影响  

Changes of M1/M2 macrophages polarization associated with active pulmonary tuberculosis and the effect of ESAT6 on macrophage polarization

作　　者：盖林林 孙维策 褚锦锦 徐栋花 GAI Linlin;SUN Weice;CHU Jinjin;XU Donghua(Central Laboratory,Weifang People′s Hospital,Weifang 261000,Shandong,China;不详)

机构地区：[1]潍坊市人民医院中心实验室,山东潍坊261000 [2]潍坊市中医院血管外科,山东潍坊261000

出　　处：《实用医学杂志》2024年第20期2867-2873,共7页The Journal of Practical Medicine

基　　金：山东省自然科学基金项目(编号:ZR2020KC001);潍坊市卫生健康委员会项目(编号:WFWSJK-2021-014)。

摘　　要：目的探讨活动性肺结核患者外周血中单核-巨噬细胞M1/M2极化的变化及结核分枝杆菌ESAT6对人源THP-1细胞极化的影响。方法收集14例活动性肺结核(活动性肺结核组)和10例健康人(健康对照组)肝素钠抗凝全血和血清,采用淋巴细胞液分离肝素钠全血中单个核细胞(PBMCs),通过实时-定量PCR仪检测确诊的活动性肺结核患者PBMCs中HLA-DR、CD11C、CD68、CD206、Arg-1的mRNA水平,应用流式细胞仪检测细胞因子(IL-2、IL-6、TNF-α、IFN-γ、IL-4等)的分泌情况。将人源THP-1细胞经佛波酯(PMA)诱导分化,将其变成巨噬细胞样细胞后,分为M0组、M1组、M2组、M0+ESAT6组,刺激24 h后,荧光定量PCR检测HLA-DR、CD11C、CD68、CD206、Arg-1的mRNA水平。ESAT6分别刺激THP-16 h、12 h、24 h后,流式技术检测细胞培养上清中相关细胞因子的水平(IL-2、IL-6、TNF-α、IL-4等)。结果与健康对照组相比,活动性肺结核组外周血PBMCs中M1极化表型分子HLA-DR、CD11C、CD68的mRNA表达水平均上调(P<0.05);M2极化表型分子CD206 mRNA的表达水平下降(P<0.05),Arg-1 mRNA的表达水平差异无统计学意义(P>0.05)。活动性肺结核组患者血清M1极化相关的促炎性细胞因子IL-2、IL-6、TNF-α、IFN-γ水平均上升(P<0.05),抗炎性细胞因子IL-4水平降低(P<0.05)。体外诱导THP-1巨噬细胞分化为不同表型,细胞M1极化表型分子HLA-DR mRNA表达水平在各组之间有统计学意义(F=21.83,P=0.000),两两比较结果显示,M1表型组、M0+ESAT6表型组均较M0组明显上调(P<0.05),其余组间差异无统计学意义(P>0.05);但CD68 mRNA表达水平在各组之间差异无统计学意义(F=2.480,P=0.135)。细胞M2极化表型分子CD206、Arg-1 mRNA水平在各组之间差异无统计学意义(F=1.233,P=0.3597;F=6.059,P=0.068)。M1极化相关的促炎性细胞因子IL-2及抗炎性细胞因子IL-4在各组细胞培养不同时间节点均差异无统计学意义(P>0.05)。与M0和ESAT6表型组相比,M1表型组促�Objective To investigate the alteration of M1/M2 polarization of monocyte-macrophages from the peripheral blood of patients with active pulmonary tuberculosis,and the effect of Mycobacterium tuberculosis ESAT6 on the polarization of human THP-1 cells.Methods Whole blood and serum samples were collected from 14 patients with active pulmonary tuberculosis and 10 healthy controls.Peripheral blood mononuclear cells(PBMCs)were isolated from whole blood with heparin sodium using lymphocyte fluid.The mRNA levels of HLA-DR,CD11C,CD68,CD206 and Arg-1 in PBMCs from patients with active pulmonary tuberculosis were detected by real-time quantitative PCR.The secretion of cytokines(IL-2,IL-6,TNF-α,IFN-γ,IL-4,etc.)was detected by flow cytometry.Human THP-1 cells were induced by phorbol ester(PMA)to differentiate into macrophages-like cells,which were divided into M0 group,M1 group,M2 group,and M0+ESAT6 group.After 24 hours of stimulation,the mRNA levels of HLA-DR,CD11C,CD68,CD206 and Arg-1 were detected by real-time PCR.Following stimulation with ESAT6 for 6 h,12 h and 24 h,the levels of cytokines(IL-2,IL-6,TNF-α,IL-4,etc.)in cell culture supernatant from THP-1 cells were detected by flow cytometry.Results Compared with the healthy control group,the mRNA expression levels of M1-polarized phenotypic molecules HLA-DR,CD11C and CD68 in PBMCs of the active pulmonary tuberculosis group were up-regulated(P<0.05).The mRNA expression level of M2-polarized phenotype molecule CD206 was decreased(P<0.05),while the expression level of Arg-1 mRNA was not statistically significant(P>0.05).Serum levels of M1-related proinflammatory cytokines IL-2,IL-6,IFN-γand TNF-αwere increased in patients with active pulmonary tuberculosis(all P<0.05),whereas decreased level of anti-inflammatory cytokine IL-4(P<0.05)were found in serum samples from patients with active pulmonary tuberculosis.THP-1 macrophages were induced to differentiate into different phenotypes in vitro,and the HLA-DR mRNA expression level of cell M1 polarization phenotype molecule

关 键 词：结核分枝杆菌 巨噬细胞 极化 活动性肺结核 6KD早期分泌靶向蛋白 

分 类 号：R521[医药卫生—内科学]