痰液miR-155检测作为诊断结核分枝杆菌感染标志物的研究  

Sputum miR-155 as a biomarker in detection with Mycobacterium tuberculosis infection

作　　者：陈思达[1] 卓宋明[1] 李娜[1] 黄震[1] 季乐财 唐晓磊 CHEN Sida;ZHUO Songming;LI Na;HUANG Zhen;JI Lecai;TANG Xiaolei(Department of Respiratory and Critical Care Medicine,Longgang District Center Hospital of Shenzhen,Shenzhen,Guangdong 518000,China;Shenzhen Center for Chronic Disease Control,Shenzhen,Guangdong 518000,China;Basic Medical Laboratory,the Second Affiliated Hospital of Wannan Medical College,Wuhu,Anhui 241000,China)

机构地区：[1]深圳市龙岗中心医院呼吸与危重症医学科,广东深圳518000 [2]深圳市慢性病防治中心,广东深圳518000 [3]皖南医学院第二附属医院基础实验室,安徽芜湖241000

出　　处：《中国热带医学》2020年第4期359-363,共5页China Tropical Medicine

基　　金：国家自然科学基金(No.81601806);广东省深圳市科技计划项目(No.JCYJ20160428145728055);广东省深圳市卫生计生系统科研项目(No.SZXJ2017035);广东省深圳市龙岗区科技发展资金(No.20160607092939550)。

摘　　要：目的分析结核分枝杆菌H37Rv感染诱导宿主巨噬细胞表达miRNA的变化,进而验证差异miRNA对结核分枝杆菌感染的临床诊断意义。方法使用H37Rv菌株感染人巨噬细胞系THP-1细胞后,收集12 h、24 h和48 h细胞并提取总RNA,进行miRNA微阵列芯片分析;再将临床分离的120株不同个体来源的结核分枝杆菌菌株与THP-1细胞共培养,使用实时定量PCR(real time-PCR,rt-PCR)验证差异miRNA的表达水平;随后收集结核患者的痰液标本处理后,提取总RNA,进行实时荧光定量PCR检测痰液中的差异表达的miRNA水平。结果H37Rv体外刺激THP-1细胞,使用miRNA微阵列芯片分析,结果显示:miR-155、miR-29b-1*、miR-150、miR-146a、miR-212和miR-483-5p共计6个miRNAs水平在3个时间点升高均较为明显;将来自不同区域的120株结核菌株分别与THP-1细胞的共培养24 h,检测发现miR-155表达水平最高;以miRNA-155为检测靶标,对68例结核患者的痰液进行检测,灵敏度和特异度分别为94.1%和93.8%。其灵敏度高于痰涂片的22.1%和结核抗体的83.8%,差异具有统计学意义(P<0.05)。但与ELISpot法的97.1%,差异无统计学意义(P>0.05)。在对痰涂片阳性结果与miRNA检测结果相关性分析中,发现miR-155其表达水平与痰涂片阳性等级呈正相关(R2=0.743,P<0.05)。结论此研究提示miR-155能够作为活动性结核诊断的有效靶标,且对间接评估患者肺部荷菌量有重要意义。Objective To analyze Mycobacterium tuberculosis(M.tb)strain H37Rv induced differential expression miRNAs from host macrophage,and we explore the diagnosis value of M.tb infection as a biomarker in clinic.Methods The Mycobacter tuberculosis(M.tb)strain H37Rv was incubated and cultured with human macrophage line THP-1.The total RNA was extracted from the THP-1 cells in 12 h,24 h and 48 h.The miRNA of groups was detected by miRNA microarray chips and the differential expressed miRNAs were analyzed for verification experiments furtherly.The rt-PCR was used to detect the differential expressed miRNAs from infected THP-1 by M.tb strains,isolated from 120 clinical sputum samples,respectively.Following,the RNA was extracted from sputum sample to detect the miRNA using rt-PCR and this detecting efficiency of several methods was compared.Results The data showed the expression level of six miRNA have a significant change,that miR-155,miR-29b-1*,miR-150,miR-146a,miR-212 and miR-483-5p.The six miRNAs were detected from THP-1 cells,were infected by 120 clinical M.tb strains from different areas for 24 h.The results showed miR-155 was a higher level than others in the tests.After that the miR-155 as a biomarker for M.tb infection diagnosis,the RNA was extracted from 68 sputum samples to detect the level of miR-155.The sensitivity and specificity of miR-155 detectionwere 94.1%and 93.8%,respectively.The sensitivity was higher than that of sputum smear in 22.1%and tuberculosis antibody in 83.8%significantly(P<0.05).But there was no significant difference with ELISPOT in 97.1%(P>0.05).The ranking of sputum smear was positive correlation with miR-155 detection in sputum(R2=0.743,P<0.05).Conclusions The data suggested that miR-155 detection is a viral significance for active TB infection diagnosis and bacteria-loads evaluation indirectly as an efficiency biomarker.

关 键 词：结核分枝杆菌 微小核糖核酸 痰液 诊断 

分 类 号：R521.1[医药卫生—内科学]