巨噬细胞通过下调miR-20a诱发凋亡促进结核菌清除的机制  

Down-regulation of miR-20a triggers macrophages cell apoptosis to facilitate mycobacterial clearance

作　　者：张娟娟 贺星[2] 梁娟[1,2] 唐怡敏 欧敏[2] 叶涛生 曾常春[3] 张国良 ZHANG Juanjuan;HE Xing;LIANG Juan;TANG Yimin;OU Min;YE Taosheng;ZENG Changchun;ZHANG Guoliang(Department of Biotechnology of Medical College of Guilin, Guilin, Guangxi 541004, China;Guangdong Province New Infectious Disease Diagnosis and Treatment of Key Laboratories, Shenzhen Third People＇s Hospital, Shenzhen, Guangdong 518112, China)

机构地区：[1]桂林医学院生物技术学院,广西桂林541004 [2]深圳市第三人民医院,广东省新发传染病诊治重点实验室,广东深圳518112 [3]深圳市龙华区中心医院,广东深圳518110

出　　处：《中国热带医学》2018年第6期523-527,共5页China Tropical Medicine

基　　金：十三五传染病重大专项(No.2017ZX10103004);国家自然科学基金(No.81501714);深圳市科技计划项目(No.JCYJ20160427153348709,JCYJ20170412151620658,JCYJ201703070950030518)

摘　　要：目的探讨上调或下调巨噬细胞中miR-20a对结核菌诱导的细胞凋亡以及细菌清除的影响,鉴定结核免疫干扰新靶点。方法构建表达miR-20a-5p、miR-20a-5p-inh且含绿色荧光蛋白(GFP)的慢病毒载体,用无血清转染试剂稀释,在5μg/mL聚凝胺存在下以感染复数(MOI)为10转染人单核细胞株THP-1细胞3 h,72 h后通过流式细胞仪筛选出GFP+细胞,佛波脂(PMA)刺激24 h后分化为巨噬细胞;结核分枝杆菌灭活菌(H37Ra)或过氧化氢(H_2O_2)处理细胞后,进行荧光素PE标记的膜联蛋白(Annexin)V和7-氨基放线菌素D(7AAD)染色15 min,流式细胞术检测Annexin V(+)和Annexin V(+)7AAD(+)细胞早晚期凋亡情况,方差分析(ANOVA)和多重比较试验(Newman-Keuls)分析细胞间凋亡率的差异;H37Ra以MOI为10感染巨噬细胞30 min,磷酸盐缓冲液(PBS)洗3次后放入CO_2培养箱,3 d后收集裂解物并接种到含10%OADC增菌液的7H11琼脂平板上,37℃培养箱培养3~4周后使用标准程序重复三次计算细菌菌落数。结果成功构建miR-20a-5p、miR-20a-5p-inh慢病毒载体以及稳定促进或抑制miR-20a表达的细胞株;流式细胞术检测显示:在未刺激情况下细胞早、晚期凋亡的差异不明显,H37Ra或H_2O_2刺激后,细胞早、晚期凋亡率发生明显变化,以晚期凋亡率显著增加为主;方差分析和多重比较试验提示:在未刺激情况下过表达或抑制miR-20a,细胞凋亡率无显著差异,H37Ra或H_2O_2刺激后在miR-20a过表达时细胞凋亡率无明显变化,抑制miR-20a的表达细胞凋亡率显著增加;H37Ra感染巨噬细胞后收集细胞裂解物并接种到平板中培养,miR-20a过表达的细胞内结核菌存活率较高,抑制miR-20a表达结核菌的存活率下降。结论结核菌感染巨噬细胞后,宿主通过下调miR-20a诱发细胞凋亡进而清除结核菌。Objective To investigate the effect of up-regulation or down-regulation of miR-20a on the macrophage apoptosis induced by M. tuberculosis and bacterial eradication, and to identify the new target for tuberculosis immune interference. Methods Lentiviral vector expressing miR-20a-5p, miR-20a-5p-inh and containing green fluorescent protein （GFP） was constructed, then the lentivirus were diluted in serum free OptiMEM and transfected into human mononuclear cell line （THP-1） cell at a multiplicity of infection （MOI） of 10 for 3 h in the presence of 5 μg/mL polybrene, from which the GFP＋ cells were isolated with fluorescence activated cell sorting （FACS） after 72 h of infection in serum-containing medium for further analysis and differentiate into macrophages in the presence of phorbol 12-myristate 13-acetate （PMA, 20 ng/mL） for 24 hours; the cells were stimulated by the live attenuated strain H37Ra or treated with hydrogen peroxide （H2O2） and incubated with annexin V-PE and 7-amino actinomycin D （7AAD） for 15 min, the annexin V（＋） and annexin V（＋） 7AAD （＋） cells were defined as the number of apoptotic cells, determined by flow cytometric analysis. ANOVA/Newman-Keuls multiple comparison test was used to compare the difference of apoptosis rates among various groups; cells were infected by H37Ra for 30 min at a MOI of 10 and washed 3 times with PBS, and 3 d later, the cell lysates were collected, serial dilutions of lysates were plated on 7H11 agar plates supplemented with 10% OADC and incubated at 37 ℃ for 3–4 weeks, the number of bacterial colonies was counted in triplicate with standard procedure. Results The miR-20a-5p, miR-20a-5p-inh lentiviral vectors were successfully constructed, and the THP-1 cell line that miR-20a gene stably increased or suppressed were established; flow cytometry analysis showed that there was no obvious difference between early and late apoptosis in the absence of stimulation, the cell apoptosis changed obviously after infected by H37Ra or

关 键 词：微小RNA 细胞凋亡 结核分枝杆菌 

分 类 号：R378.981[医药卫生—病原生物学]