【作者】 张向晖；

【导师】 袁俐；

【作者基本信息】 石河子大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 结核病是一个古老的、在全球广泛流行的传染病,至今仍然是威胁人民健康的重要公共卫生问题,而且可能是一个阻碍社会和经济发展的社会问题。自1882年Koch氏发现结核分支杆菌以来,全球己有2亿人死于结核病,现在全球约20亿的人口受到结核菌感染,结核病例有2000万。乙胺丁醇(EMB)作为一种重要的抗结核化疗药物,对增殖期的结核分支杆菌有选择性抑菌作用,自从应用于抗结核治疗以来,疗效一直较为满意。但从二十世纪八十年代中期开始,由于结核分支杆菌自身的适应性改变、不正规的治疗、移民和爱滋病的流行等原因结核分支杆菌对抗结核药物的耐药情况变得严重起来。EMB耐药情况虽然不如异烟肼(INH)、利福平(RFP)和链霉素(SM)那么严重,但近来亦有不断升高的趋势。近年来,不少临床和科研人员一直致力于EMB耐药机制的研究,但到目前为止对EMB的作用机制和耐药机制仍然没有完全清楚。本研究通过对EMB耐药结核分枝杆菌embB基因片段的分析,了解我国新疆部分地区结核分支杆菌EMB耐药和embB基因突变的关系,并进一步探索快速检测结核分支杆菌EMB耐药性的实验方法。目的:本文阐述了分支杆菌耐乙胺丁醇(EMB)的分子机制,了解了结核杆菌耐EMB分离株embB基因突变情况;探索了聚合酶链反应一单链构象多态性(PCR-SSCP)分析技术检测结核杆菌耐乙胺丁醇的应用价值,建立了快速、有效的结核分枝杆菌药物敏感性试验方法。方法:临床标本进行传统分支杆菌培养、菌种鉴定和药敏试验;通过PCR-SSCP和PCR- RFLP技术分析结核分支杆菌临床分离株embB基因。结果:对98株结核分支杆菌进行了传统药物敏感性试验,52株为EMB敏感株,46株为耐EMB菌株。从Genbank下载结核分支杆菌embB基因序列,应用引物设计软件Premier5.0设计一对引物XH1和XH2扩增embB基因的400bpDNA片段。以结核分支杆菌标准菌株H37Rv做对照,98株结核分支杆菌临床分离株embB基因400bp扩增片段经限制性内切酶HaeⅢ酶切后,结果显示:52株EMB敏感株和39株耐EMB菌株的PCR-限制性片段长度多态性(RFLP)图谱与结核分支杆菌标准株相同,余7株耐EMB菌株RFLP图谱与结核分支杆菌标准株有明显差异。通过PCR-SSCP方法分析98株结核分支杆菌分离株embB 400bp片段,发现52株EMB敏感株SSCP图谱与结核分支杆菌标准菌株相同;46株耐EMB菌株中,28株(60.9%)SSCP图谱与结核分支杆菌标准株不同,余18株SSCP图谱与结核分支杆菌标准株相同。结论:对引物XH1和XH2扩增的400bp片段,采用8%(29:1)非变性聚丙烯酞胺凝胶在150V稳压下电泳7小时效果较理想。embB基因突变,尤其306位密码子突变是结核分支杆菌耐乙胺丁醇的主要分子机制。PCR-SSCP可快速检测出部分结核分支杆菌耐EMB临床分离株,但比DNA测序简便、快速,便于临床推广应用。更多还原

【Abstract】 Mycobacterium tuberculosis was a widely transmission disease with a history and threatening human health and social development. Since Koch found M.tuberculosis in 1882, 200 million died of M.tb over world. Now,2 billion were infected with M.tb and 20 million were M.tb patients.Ethambutol(EMB) is one of the most important anti-tuberculosis drugs; it can selectively inhibit the growth of Mycobacterium tuberculosis, which is in the reproductive period. Since it has been used in the treatment of tuberculosis, EMB has been proved to be an effective anti-tuberculosis agent. In recent twenty years, multi-drug resistance of Mycobacterium tuberculosis has become a serious problem due to the adaptive change of Mycobacterium tuberculosis itself, inappropriate therapy, the prevalence of AIDS, and other reasons. Although the condition of drug resistance to EMB is not so serious as that of the resistances to INH, SM, and RFP, there is a rising tendency of the EMB-resistance rate to Mycobacterium tuberculosis. Recently, many clinicians and researchers devoted themselves to the study of the mechanism of EMB resistance, but the acting mechanism and resistant mechanism of EMB are still not very clear now.In this study we want to analyze the relationship between EMB-resistance and the embB gene mutation in part area of Xinjiang Province in China, and develop a rapid method to screen drug resistant bacterium.Objective: To expound molecular mechanism of ethambutol resistance, and understand the mutations of embB genes in M.tuberculosis isolates; To explore the applying value that Polymersae chain reaction-Single stranded conformation polymorphism(PCR-SSCP) detect ethambutol resistance in M.tuberculosis, and establish a rapid, valid drug susceptibility test for genoytpes of M.tuberculosis EMB-resistance.Methods: Traditional culture, species identification of mycobacteria and drug susceptibility testing were performed to clinical specimens. embB gene of clinical isolates in M.tuberculosis was analyzed by PCR-SSCP and PCR-restriction fragment length polymorphism(RFLP).Results: 98 isolates of M.tuberculosis were performed by traditional drug susceptibiliyt testing, 52 EMB-susceptible and 46 EMB-resistant isolates were showed.The 400bp fragment of embB gene was amplified by primers XH1 and XH2, which are designed with the software Premier 5.0 according to the sequence information downloaded from genbank database.We used the standard strain of M.tuberculosis as control group, 98 clinical isolates of M.tuberculosis were digested by restriction endonuclease HaeⅢ, the results indecated: the characterization of RFLP in 52 EMB-susceptible and 39 EMB-resistant clinical isolates was similar to standard strain, 7 EMB-resistant isolates had abnormal RFLP profiles. We analyzed the amplified products of the 98 isolates by PCR-SSCP, among 46 EMB-resistant M.tuberculosis isolates, the profiles of SSCP in 28 isolates were diferent from that of the standard strain, no diference was found in 52 EMB-susceptible and 18 EMB-resistant isolates. Conclusion: The testing effects were more ideal to embB 400bp fragments when 8% gel concentration (29:1) was used under the action of 150 voltage for 7 hours.The mutations at codon 306 of embB were important molecular mechanism in EMB-resistance of M.tuberculosis. PCR-SSCP can rapidly detect the EMB-resistant isolates of M.tuberculosis. The data were identical compared to DNA sequencing, but PCR-SSCP had the advantages such assimple, rapid, and suitable for clinical apply.更多还原