转染miR-223模拟物的巨噬细胞结核分枝杆菌清除能力、凋亡、炎症反应变化及miR-223与NLRP3靶向关系  

Changes in the ability to clear Mycobacterium tuberculosis,apoptosis,inflammatory response of mac⁃rophages transfected with miR-223 mimics and the targeted relationship between miR-223 and NLRP3

作　　者：李达[1] 刘波[1] 刘丽红[1] 李秋平 LI Da;LIU Bo;LIU Lihong;LI Qiuping(Department of Laboratory,Qinhuangdao First Hospital,Qinhuangdao 066000,China)

机构地区：[1]秦皇岛市第一医院检验中心,河北秦皇岛066000

出　　处：《山东医药》2024年第24期33-37,共5页Shandong Medical Journal

基　　金：河北省医学科学研究重点课题计划项目(20201317)。

摘　　要：目的 观察肺结核患者血清微小RNA-223(miR-223)水平变化,以及转染miR-223模拟物的巨噬细胞结核分枝杆菌(MTB)H37Rv清除能力、凋亡、炎症反应变化,并分析miR-223与NLRP3靶向关系。方法 选取37例份活动性肺结核患者血清标本(肺结核组)和同期40例健康体检者血清标本(健康组),采用RT-PCR法检测两组血清标本中miR-223。体外培养人单核细胞株THP-1,经佛波脂刺激24 h后分化为巨噬细胞,将巨噬细胞随机分为健康组、MTB组、MTB+mimics-NC组、MTB+miR-223 mimics组,健康组细胞常规培养,MTB组、MTB+mimics-NC组、MTB+miR-223 mimics组均用MTB标准株H37Rv感染巨噬细胞,感染后MTB+mimics-NC组和MTB+miR-223 mimics组应用脂质体转染法分别将mimics-NC、miR-223 mimics转染至细胞中,采用RT-PCR法检测各组细胞miR-223及NLRP3、TNF-α、IFN-γ mRNA,流式细胞术测算各组细胞凋亡率,菌落形成单位(CFU)计数法检测各组H37Rv细菌负荷量。取对数生长期THP-1细胞,随机分为miR-223 mimics+NLRP3-WT组、mimics-NC+NLRP3-WT组、miR-223mimics+NLRP3-MUT组、mimics-NC+NLRP3-MUT组,用Lipofectamine 2000按照试剂盒说明书操作分别将miR-223mimics和NLRP3-WT、mimics-NC和NLRP3-WT、miR-223 mimics和NLRP3-MUT、mimics-NC和NLRP3-MUT共转染至各组细胞,转染后24 h检测各组细胞的相对荧光素酶活性,双荧光素酶报告基因实验验证miR-223与NLRP3是否存在靶向关系。结果 与健康组比较,肺结核组血清miR-223表达降低(P<0.05)。与健康组比较,MTB组miR-223表达、细胞凋亡率降低,NLRP3、TNF-α、IFN-γ mRNA表达及H37Rv细菌负荷量升高(P均<0.05);与MTB+mimics-NC组比较,MTB+miR-223 mimics组miR-223表达、细胞凋亡率升高,NLRP3、TNF-α、IFN-γ mRNA表达及H37Rv细菌负荷量降低(P均<0.05)。双荧光素酶报告基因实验显示,NLRP3是miR-223的靶向基因。结论 肺结核患者血清miR-223表达降低,转染miR-223模拟物能够促进巨噬细胞凋亡,增强MTB感染的巨噬细胞Objective To observe the changes in serum microRNA-223(miR-223)levels in patients with pulmo-nary tuberculosis,as well as the changes in the clearance ability to clear Mycobacterium tuberculosis(MTB)H37Rv,apoptosis and inflammatory response of macrophages transfected with miR-223 mimics,and to analyze the targeted relation-ship between miR-223 and NLRP3.Methods Thirty-seven serum samples from active pulmonary tuberculosis patients(tuberculosis group)and 40 serum samples from healthy individuals during the same period(healthy group)were select-ed,and miR-223 was detected in the serum samples of both groups using RT-PCR.Human monocyte line THP-1 was cul-tured in vitro and differentiated into macrophages after 24 h of stimulation with phorbol.Macrophages were randomly divid-ed into the healthy group,MTB group,MTB+mimics-NC group,and MTB+miR-223 mimics group.Cells in the healthy group were cultured routinely,while cells in the MTB group,MTB+mimics-NC group,and MTB+miR-223 mimics group were all infected with MTB standard strain H37Rv.After infection,in the MTB+mimics-NC group and MTB+miR-223 mimics group,we used liposome transfection method to transfect mimics-NC and miR-223 mimics into cells,respective-ly.The miR-223,NLRP3,TNF-αand IFN-γmRNA in cells of each group were detected by RT-PCR.Flow cytometry was used to detect the apoptosis rate of cells in each group,and colony forming unit(CFU)counting method was used to detect the H37Rv bacterial load in each group.THP-1 cells in the logarithmic growth phase were taken and randomly di-vided into the miR-223 mimics+NLRP3-WT group,the mimics-NC+NLRP3-WT group,the miR-223 mimics+NLRP3-MUT group,and the mimics-NC+NLRP3-MUT group,respectively.The miR-223 mimics and NLRP3-WT,mimics-NC and NLRP3-WT,miR-223 mimics and NLRP3-MUT,and mimics-NC and NLRP3-MUT were co-transfected into cells of the above groups,respectively,by Lipofectamine 2000 reagent in accordance with the operation instructions of the kit.The relative luciferase activity of cells in each group was detected at 2

关 键 词：微小RNA-223 结核分枝杆菌 巨噬细胞 肺结核病 活动性肺结核病 NOD样受体热蛋白结构域相关蛋白3 

分 类 号：R378[医药卫生—病原生物学]