本文選題：結(jié)核病　切入點(diǎn)：復(fù)發(fā)　出處：《復(fù)旦大學(xué)》2006年博士論文

【摘要】： 結(jié)核病是一種古老的傳染性疾病，在歷史上曾經(jīng)奪取數(shù)以萬計(jì)的生命。近年來由于全球人口流動的加速，人類免疫缺陷病毒感染的流行，耐藥菌株的出現(xiàn)和傳播，以及其它一些經(jīng)濟(jì)和政策等方面的原因，使得結(jié)核病卷土重來，再次構(gòu)成對人類健康的重大危害。我國的結(jié)核病控制工作盡管已經(jīng)取得了很大的進(jìn)步，但是形勢依然很嚴(yán)峻。結(jié)核病的復(fù)發(fā)已成為我國結(jié)核病實(shí)際防治工作中面臨的難題。本研究根據(jù)我國較高的結(jié)核病復(fù)發(fā)率，選擇上海地區(qū)的復(fù)發(fā)病人作為研究對象，利用基因型分型技術(shù)，對復(fù)發(fā)的病因?qū)W進(jìn)行了研究。同時(shí)，針對復(fù)發(fā)患者中較高的耐藥率，在文獻(xiàn)綜述的基礎(chǔ)上，對一線抗結(jié)核藥物乙胺丁醇(ethambutol，EMB)特殊的耐藥現(xiàn)象及其耐藥分子機(jī)制進(jìn)行了探索性研究。全文分二個(gè)部分： 第一部分結(jié)核病復(fù)發(fā)的病因?qū)W研究 為研究結(jié)核病復(fù)發(fā)的病因?qū)W，首先評估了一種新的結(jié)核分枝桿菌基因型分型法—分枝桿菌散在分布重復(fù)單位基因型分析法(MIRU)。隨機(jī)抽取上海地區(qū)2000年至2002年分離保存的91株菌株，對其進(jìn)行MIRU分型。發(fā)現(xiàn)91株菌株用該分型法可得到46種基因型。12個(gè)MIRU位點(diǎn)的多態(tài)性分析表明各位點(diǎn)之間有較大的區(qū)別，其中位點(diǎn)26顯示了較高的多態(tài)性，位點(diǎn)16、31、40顯示了中等程度的多態(tài)性。與另一種以PCR為基礎(chǔ)的基因型分析技術(shù)Spoligotyping相比，MIRU顯示了較好的分辨率。進(jìn)一步以上海地區(qū)1999年至2004年復(fù)發(fā)肺結(jié)核患者為對象，，通過比較復(fù)發(fā)患者前后兩次發(fā)病時(shí)結(jié)核分枝桿菌MIRU基因型的差異，來判斷結(jié)核病復(fù)發(fā)的真正原因。在52例結(jié)核病復(fù)發(fā)患者中，32例患者兩次發(fā)病時(shí)結(jié)核分枝桿菌的MIRU基因型發(fā)生了變化，提示62％的結(jié)核病患者的復(fù)發(fā)是由于外源性再感染而引起的。隨著年齡的增加，由外源性再感染所致復(fù)發(fā)的可能性逐步降低，≤30歲、31～60歲、≥60歲復(fù)發(fā)患者中外源性再感染的比例分別為100％(4／4)、67％(12／18)、53％(16／30)。隨著復(fù)發(fā)間隔時(shí)間的延長，結(jié)核病復(fù)發(fā)患者中由外源性再感染所致的可能性逐步增加，6個(gè)月內(nèi)復(fù)發(fā)的患者中47％(7／15)是由外源性再感染引起的，而1年以后這個(gè)比例達(dá)到74％(17／23)。復(fù)發(fā)患者中較高的外源性再感染比例提示結(jié)核病近期傳播現(xiàn)象較嚴(yán)重，結(jié)核病防治工作中應(yīng)當(dāng)加強(qiáng)傳染源的早期發(fā)現(xiàn)，切斷傳播途徑。外源性再感染在結(jié)核病患者中常見，也說明人體免疫系統(tǒng)缺乏有效的保護(hù)機(jī)制，不能保護(hù)機(jī)體免受結(jié)核分枝桿菌再次感染，這對結(jié)核病預(yù)防性疫苗的研究和開發(fā)提出了新的挑戰(zhàn)。 第二部分結(jié)核分枝桿菌EMB耐藥的分子機(jī)制研究 EMB是一種目前普遍采用的一線抗結(jié)核藥物。先前的研究發(fā)現(xiàn)乙胺丁醇耐藥常常與其它耐藥聯(lián)系在一起，為了觀察這種現(xiàn)象是否與不同地區(qū)的菌株流行相關(guān)，本研究對上海市10年來保存的菌株的藥敏結(jié)果進(jìn)行了分析。結(jié)果表明，單耐EMB的菌株數(shù)顯著少于單耐其它常用抗結(jié)核藥物(異煙肼、利福平和鏈霉素)的菌株數(shù)。在耐兩種以上藥物的菌株中，耐EMB的菌株數(shù)量與菌株耐藥種類的數(shù)目成正相關(guān)，提示菌株耐EMB與耐其它抗結(jié)核藥物存在相關(guān)性。為研究embB306位點(diǎn)突變與菌株耐藥的關(guān)系，本研究分析了116株耐藥菌株和54株全敏感菌株embB306位點(diǎn)的突變情況。結(jié)果發(fā)現(xiàn)，在所有耐EMB的27株菌株中，共檢測到embB306位點(diǎn)突變17株，在不耐EMB但耐其它藥物的89株菌株中也檢測到突變17株，在全敏感菌株中沒有檢測到embB306位點(diǎn)的突變。經(jīng)統(tǒng)計(jì)學(xué)檢驗(yàn)，embB306突變不僅與EMB耐藥之間存在相關(guān)性，也與耐其它常用抗結(jié)核藥物存在相關(guān)性。同時(shí)也發(fā)現(xiàn)embB306突變所占的比例隨著耐藥種類的增加而增加。由于臨床上EMB單獨(dú)耐藥菌株數(shù)目較少，因而檢測embB306位點(diǎn)的突變對檢測菌株EMB單獨(dú)耐藥的實(shí)際意義不大。但embB306位點(diǎn)突變與菌株耐多藥之間存在一定的相關(guān)性，因而檢測該位點(diǎn)的突變可以作為快速篩查臨床耐多藥菌株的候選分子標(biāo)記。 結(jié)核分枝桿菌產(chǎn)生耐藥的機(jī)制與其它細(xì)菌略有不同，質(zhì)粒、轉(zhuǎn)座子以及整合子等以基因水平傳播方式介導(dǎo)的耐藥機(jī)制較少見，染色體上的缺失和突變被認(rèn)為是其產(chǎn)生耐藥的主要方式。為進(jìn)一步深入理解分枝桿菌EMB耐藥產(chǎn)生的分子機(jī)制，以恥垢分枝桿菌作為研究模型，采用轉(zhuǎn)座子突變技術(shù)研究與EMB耐藥相關(guān)的新基因。使用基于Himar1轉(zhuǎn)座子的MycoMarT7轉(zhuǎn)座子系統(tǒng)，構(gòu)建了恥垢分枝桿菌轉(zhuǎn)座子突變文庫。Southern blot實(shí)驗(yàn)證實(shí)Himar1轉(zhuǎn)座子以隨機(jī)單拷貝插入的方式分布在染色體上。進(jìn)一步對該文庫進(jìn)行EMB耐藥篩選，分離獲得了4個(gè)耐藥突變株分別命名為ER2A、ER4A、ER5A和ER7A，經(jīng)MIC測定其耐藥能力比野生型提高了20倍以上。使用接頭PCR和標(biāo)記挽救方法鑒定了各突變株中轉(zhuǎn)座子在基因組中的插入位點(diǎn)。生物信息學(xué)分析表明，ER2A突變菌株中轉(zhuǎn)座子插入部位基因編碼單加氧酶，該酶可能參與分枝桿菌中間產(chǎn)物的代謝；ER4A突變株插入部位的基因?yàn)閘prE，編碼一種細(xì)胞內(nèi)的脂蛋白；ER5A突變株插入部位的基因?yàn)榫幋a轉(zhuǎn)運(yùn)蛋白的arsA；ER7A突變株中插入部位編碼的基因功能未知。這些新分離到基因耐乙胺丁醇的機(jī)制有待進(jìn)一步深入研究。
[Abstract]:Tuberculosis is an infectious disease of old, in history has killed millions of people. In recent years due to the acceleration of global population, the prevalence of human immunodeficiency virus infection, the emergence and spread of resistant strains, and other economic and policy issues, the TB comeback again pose a significant harm to human health our work in tuberculosis control. Although great progress has been made, but the situation is still very serious. The recurrence of tuberculosis has become a problem for the actual work of prevention and control of tuberculosis in China in this study. According to our country's higher recurrence rate of tuberculosis in Shanghai area, the recurrence of patients as the research object, using gene typing technique the etiology of recurrence, were studied. At the same time, the drug resistance in patients with high recurrence rate, on the basis of literature review, on a The special drug resistance of ethambutol (EMB) and its molecular mechanism were studied. The full text is divided into two parts:
The etiological study of the first part of tuberculosis recurrence
As the etiology of recurrent tuberculosis, first evaluate a new Mycobacterium tuberculosis genotyping method - mycobacterial interspersed repetitive unit genotype analysis (MIRU) were randomly selected in Shanghai. From 2000 to 2002 from the collection of 91 strains were genotyped by MIRU. 91 strains with the classification method can obtain polymorphism analysis of 46 genotypes of.12 MIRU loci showed a larger difference between you, the site 26 showed high polymorphism loci, 16,31,40 showed moderate polymorphism. Compared with another PCR based genotyping technology Spoligotyping, MIRU show good resolution. Further in Shanghai from 1999 to 2004 the recurrence of pulmonary tuberculosis patients as the object, by comparing the recurrence of patients before and after two times at the onset of Mycobacterium tuberculosis MIRU gene type, to determine the node The real reason for the nuclear attack. In 52 cases of recurrent tuberculosis patients, 32 patients changed two times at the onset of Mycobacterium tuberculosis MIRU gene, showed that 62% of the patients with tuberculosis recurrence is due to exogenous re infection. With the increase of age, the possibility of exogenous re infection caused by recurrence gradually reduced, less than 30 years, 31~60 years, more than 60 years old patients with recurrent exogenous reinfection rates were 100% (4 / 4), 67% (12 / 18), 53% (16 / 30). With prolonging recurrence interval, the possibility of recurrence in patients with tuberculosis by exogenous re infection gradually increase the recurrence within 6 months were 47% (7 / 15) is caused by exogenous infection, and 1 years later, this proportion reached 74% (17 / 23). Exogenous re infection is higher in patients with recurrent ratio that recent transmission of tuberculosis is serious, tuberculosis In the prevention and treatment should be found early strengthen the source of infection, cut off the transmission. Exogenous re infection is common in patients with tuberculosis, also shows that the lack of effective protection mechanisms of the human immune system, can protect the body against Mycobacterium tuberculosis infection again, the prevention of TB vaccine research and development has brought new challenges.
Molecular mechanism of EMB resistance in the second part of Mycobacterium tuberculosis
EMB is a widely used first-line anti tuberculosis drugs. Previous studies found that ethambutol resistance and other resistant often together, in order to observe whether this phenomenon is related with the prevalent strains in different regions, the study of drug sensitive strains in Shanghai city for 10 years to save the results were analyzed. The results showed that the bacteria the number of single EMB resistance is significantly less than the single resistance to other antituberculosis drugs (isoniazid, rifampicin and streptomycin) of the strains. In more than two kinds of drug resistant strains of EMB resistant strains, the number and the number of drug-resistant strains of species are positively related, suggesting that EMB resistant strains and resistant to other anti tuberculosis drugs are associated. For the study of embB306 mutation with the strain resistance, this study analyzed the mutation of 116 strains of resistant strains and 54 strains of sensitive strain embB306 loci. The results showed that in all the 27 strains resistant to EMB, EmbB306 mutations were detected in 17 strains, not resistant to EMB but resistant to other drugs among the 89 strains have mutation was detected in 17 strains, no mutation detected embB306 loci in all strains. By statistical analysis, the correlation between embB306 mutation with EMB drug resistance, and resistance to other antituberculosis drugs there is a correlation. Also found that the proportion of embB306 mutations account for the increased with the increase of resistant species. Due to the clinical EMB alone resistant strains of the small number of the detection of embB306 mutations is of practical significance to detect drug resistance strain EMB alone. But the mutation of embB306 is associated with multi drug resistant strains, and detection of the mutation sites of the candidate molecular markers can be used for rapid screening of clinical multi drug resistant strains.
Mycobacterium tuberculosis drug resistance mechanism is slightly different, with other bacterial plasmid, transposon and integron resistance mechanism to gene level communication mediated by a rare, lack of chromosome and mutation is considered the main resistance. For further understanding the molecular mechanism of Mycobacterium tuberculosis drug resistance to EMB. In Mycobacterium smegmatis as the research model, transposon mutagenesis technique and the related research of EMB resistance gene by using Himar1. The new transposon based MycoMarT7 transposon system, constructs a transposon mutant library of.Southern blot confirmed that Himar1 transposon random single copy insertion mode on the chromosomes of Mycobacterium smegmatis further. The library was EMB resistance screening, obtained 4 resistant mutants were named as ER2A, ER4A, ER5A and ER7A, determined by MIC resistant ability than the wild Type increased more than 20 times. The use of PCR markers and joint rescue method of the mutants the transposon insertion sites in the genome were identified. Bioinformatics analysis showed that ER2A mutant strains of the transposon insertion site gene encoding monooxygenase, this enzyme may be involved in mycobacterial intermediate metabolism; ER4A insertion mutant part of the gene is lprE, encoding an intracellular lipid protein; ER5A mutant gene encoding the insertion site for transporter arsA; ER7A mutation gene encoding unknown function insertion site lines. These new isolated resistance gene B ETHAMBUTOLE mechanism needs further research.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R378;R52

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 鄧艷琴;結(jié)核分枝桿菌基因分型及異煙肼新耐藥突變位點(diǎn)的確認(rèn)研究[D];福建醫(yī)科大學(xué);2009年

相關(guān)碩士學(xué)位論文 前2條

1 張?jiān)封?環(huán)介導(dǎo)恒溫?cái)U(kuò)增技術(shù)檢測結(jié)核分枝桿菌和超級細(xì)菌的研究[D];吉林大學(xué);2012年

2 李學(xué)剛;肺結(jié)核患者愈后健康知識水平及行為對比分析[D];青島大學(xué);2013年

本文編號：1707941