即时痰和体外培养条件下结核分枝杆菌sigma因子表达差异分析  

Differential expression of sigma factor of Mycobacterium tuberculosis under in vivo infection and in vitro culture conditions

作　　者：江丽娜[1] 穆成[1] 孙蕊[1] 王春花[1] JIANG Lina;MU Cheng;SUI Rui;WANG Chunhua(Tuberculosis Reference Laboratory,Tianjin Center for Tuberculosis Control,Tianjin 300041,China)

机构地区：[1]天津市结核病控制中心,天津300041

出　　处：《公共卫生与预防医学》2023年第3期52-55,共4页Journal of Public Health and Preventive Medicine

基　　金：2017年度结核病控制中心科技基金项目(津疾控科[2013])。

摘　　要：目的为了解sigma因子在即时痰的表达情况,探索与致病性结核分枝杆菌毒力关系密切的sigma因子。方法选取2018年1~12月天津市结核病控制中心门诊部确诊的结核病患者,对其痰涂片阳性和xpert阳性确诊的肺结核随机选择20份痰阳性标本进行实验。每一例肺结核患者在其开始治疗前收集2份即时痰标本,一份立刻抽提RNA,一份进行体外培养。体外培养的菌株在生长对数期提取RNA。设计13个sigma因子的特异性引物,通过荧光定量PCR(RT-PCR)的方法,分析其在即时痰临床分离株和体外培养株的转录表达差异。以核糖体16s为参照基因,利用2^(ΔCt)分析sigma因子的转录倍数,将稳定表达的sigA作为统计分析的底物,单因素方差ANOVA分析其余sigma因子的表达差异。结果临床分离菌与H37Rv相比,所有的sigma因子统计分析差异均不显著(P均>0.05);临床分离菌与体外培养株相比,只有sigM表达量最高(P<0.05),统计分析差异显著。结论sigM在细菌感染的初级阶段转录水平明显增高,但其具体作用还不清楚,可能参与了宿主免疫系统和细菌逃离机制之间的相互平衡。Objective To investigate the gene expression of sigma factors in vivo,and to explore the sigma factors that may be closely related to the virulence of pathogenic Mycobacterium tuberculosis Methods Tuberculosis(TB)patients diagnosed in the outpatient department of Tianjin Tuberculosis Control Center from January to December 2018 were selected,and 20 sputum-positive specimens were randomly selected from TB patients confirmed with Xpert-positive for the present study.Two immediate sputum specimens were collected from each case of pulmonary tuberculosis before treatment,one for RNA extraction and one for in vitro culture.In vitro cultured strains in the logarithmic phase of growth were harvested for RNA extraction.The specific primers for 13 sigma factors were designed.The differential expression of the 13 sigma factors between sputum isolates and in vitro cultured strains was analyzed by fluorescence quantitative PCR.Taking ribosomal 16s as the reference gene,the transcription level of sigma factors was analyzed by 2^(ΔCt).Using the stably expressed sigA as the control reference,the expression differences of other sigma factors were analyzed by one-way ANOVA.Results Within 0 days,stress-associated sigma factors have a different expression profile in clinical isolate strains vs H37Rv or in vitro.All the sigma factors induced up regulation in sputum,while no difference transcription between clinical isolate strains vs H37Rv(P>0.05).When compared to in vitro culture,only sigM transcript highest in sputum(P<0.05).Conclusion SigM plays an important role in the initial stages of bacterial infection,but its exact role is unclear.We assumed it could have a role in the interplay between the host immune defenses and the bacterial escape mechanisms.

关 键 词：结核分枝杆菌 sigma因子 痰液 荧光定量PCR(RT-PCR) 

分 类 号：R181[医药卫生—流行病学]