本文選題：幽門(mén)螺桿菌　切入點(diǎn)：微進(jìn)化　出處：《中國(guó)疾病預(yù)防控制中心》2015年博士論文

【摘要】：幽門(mén)螺桿菌(Helicobacter pylori,簡(jiǎn)稱H. pylori或HP)是引起慢性胃炎、消化性潰瘍和胃癌的主要病原體。HP與特發(fā)性血小板減少性紫癜、缺血性貧血等胃腸道外疾病的相關(guān)性也日益受到重視。前期研究發(fā)現(xiàn)腎小球腎炎患者的腎臟組織存在可以與HP抗體結(jié)合的抗原,HP感染可能參與腎炎的發(fā)生發(fā)展。由于人群中存在較多比例的多株HP混合感染,獲得真正的關(guān)聯(lián)菌株用于致病性研究非常重要。目前,HP根除率顯著下降,特別是在包含阿莫西林(amoxicillin, AMO)的治療方案中,不能完全用質(zhì)子泵抑制劑(proton pump inhibitor, PPI)選擇和克拉霉素等抗生素的耐藥性升高來(lái)解釋。雖然AMO的總體耐藥率較低,但AMO用藥對(duì)HP根除治療中常用抗生素的總體敏感性的影響缺乏研究。第一部分：某腎炎患者幽門(mén)螺桿菌感染菌株微進(jìn)化及相關(guān)分析本研究利用多位點(diǎn)序列分析技術(shù)、基因組測(cè)序以及耐藥表型的檢測(cè),對(duì)來(lái)自1例慢性腎炎患者的18個(gè)HP克隆的遺傳特征、群體結(jié)構(gòu)關(guān)系及微進(jìn)化過(guò)程進(jìn)行了詳細(xì)分析。研究結(jié)果顯示該患者感染的HP是源于不同祖先菌株的兩個(gè)克隆譜系的混合感染。兩個(gè)祖先菌株至少在12年前就開(kāi)始了共感染過(guò)程,結(jié)果顯示出了在患者胃內(nèi)分別經(jīng)歷的2.8年和4.2年的微進(jìn)化,并形成各自遺傳多樣性特征的兩個(gè)克隆譜系的過(guò)程�？寺∽V系間連續(xù)的重組導(dǎo)致一定程度的趨同進(jìn)化。兩個(gè)克隆譜系重組率相差10倍,并帶有明顯不同的限制-修飾系統(tǒng)。首次直接在臨床分離株中檢測(cè)到含有原受體菌的散在序列(interspersed sequences of the recipient, ISR)的重組片段,并發(fā)現(xiàn)了噬菌體頻繁感染導(dǎo)致的重組事件,提示噬菌體在HP進(jìn)化中發(fā)揮重要作用�；蚪M比較發(fā)現(xiàn)兩個(gè)克隆譜系雖然均具有完整的Cag毒力島和相同序列的Vac A,但兩克隆系間存在50 kb差異,該區(qū)編碼完整的2型轉(zhuǎn)座子TnPZ,為進(jìn)一步開(kāi)展2型TnPZ與HP致病相關(guān)性,特別是與患者腎炎發(fā)病關(guān)系的研究提供了重要線索。第二部分：幽門(mén)螺桿菌阿莫西林耐藥相關(guān)基因表達(dá)及序列分析HP AMO耐藥分為不穩(wěn)定的表型耐藥和穩(wěn)定的遺傳耐藥兩種形式。本研究選擇AMO敏感株HP 26695作為出發(fā)菌株實(shí)驗(yàn)室誘導(dǎo)獲得AMO耐藥HP。分析不同AMO濃度下,5種青霉素結(jié)合蛋白(PBP1-PBP5)、2種孔蛋白(HopB和HopC)和一個(gè)外排泵蛋白(HefA)編碼基因的序列變化及mRNA水平的表達(dá)差異。qRT-PCR結(jié)果顯示,在不同濃度AMO的選擇壓力下,HP各檢測(cè)基因的mRNA表達(dá)水平不相同。AMO 4.0 mg/1以下(0.5、1.0、2.0 mg/1), PBP1和PBP2的mRNA表達(dá)水平均高于敏感株2倍以上,外排泵蛋白HefA的mRNA表達(dá)在AMO 0.5和1.0 mg/l濃度也呈2倍以上上調(diào)；在4.0 mg/l的AMO壓力下,則主要表現(xiàn)為PBP1～PBP3的mRNA表達(dá)上調(diào),孔蛋白HopB和HopC的mRNA表達(dá)下調(diào)；而AMO達(dá)到8.0 mg/l濃度時(shí),HopC mRNA明顯下調(diào)。以上結(jié)果說(shuō)明HP可以通過(guò)增加PBP的表達(dá)、促進(jìn)細(xì)胞壁合成,提高外排泵蛋白表達(dá)、增加藥物外排或者降低孔蛋白表達(dá)量、減少藥物攝取等多種機(jī)制提高AMO耐藥水平。本研究還獲得了AMO遺傳耐藥株,并在PBP1編碼基因新發(fā)現(xiàn)T593K和594G+兩個(gè)AMO耐藥相關(guān)的突變位點(diǎn)。本研究還發(fā)現(xiàn),單純的AMO選擇壓力,可同時(shí)造成不同程度的左氧氟沙星,莫西沙星耐藥以及高水平四環(huán)素的耐藥。以上結(jié)果表明,盡管目前AMO耐藥率低,臨床分離到穩(wěn)定耐藥株很少,但是AMO表型耐藥的存在以及因?yàn)槭褂肁MO導(dǎo)致的多重耐藥的發(fā)生,可能是HP根除率下降不可忽視的因素。以上現(xiàn)象尚未見(jiàn)相關(guān)報(bào)道。本研究通過(guò)上述宿主內(nèi)HP菌群特征和微進(jìn)化分析以及HP AMO表型耐藥和遺傳耐藥機(jī)制的研究,加深了對(duì)HP進(jìn)化和遺傳多樣性產(chǎn)生機(jī)制以及AMO耐藥機(jī)制的認(rèn)識(shí),對(duì)后續(xù)HP致病相關(guān)性研究及HP的防治具有一定的意義。
[Abstract]:Helicobacter pylori (Helicobacter pylori, referred to as H. pylori or HP) is the main pathogens of.HP cause chronic gastritis, peptic ulcer and gastric cancer and idiopathic thrombocytopenic purpura, anemia and gastrointestinal related ischemic disease has received increasing attention. Previous study found that can bind with the antibody of HP antigen in patients with glomerulonephritis kidney tissue, HP infection may be involved in the development of nephritis. Due to the presence of a higher percentage among the strains of HP mixed infection, get really very important for related strains of pathogenic research. At present, the eradication rate of HP decreased significantly, especially in containing amoxicillin (amoxicillin, AMO) of the treatment, not completely by proton pump inhibitor (proton pump inhibitor, PPI) and clarithromycin resistance selection of antibiotics increased to explain. Although the overall resistance rate of AMO was low, but AMO The lack of research on the effects of HP eradication of drug commonly used antibiotics in the treatment of the overall sensitivity. The first part: the evolution of micro strain and related multilocus sequence analysis using the analysis technology of a nephritis patients with Helicobacter pylori infection, genome sequencing and detection of drug resistance phenotype, the genetic characteristics from 18 HP clones, 1 cases of chronic nephritis patients the detailed analysis of relationship between population structure and micro evolution. The results showed that the infection of patients with HP mixed infection of two clones derived from different ancestral lineage strains. Two strains of ancestors at least 12 years before the start of the co infection process, the results show that the experience in patients with stomach 2.8 years and 4.2 years in the process of micro evolution, two clones and their pedigree forms characteristic. The genetic diversity between successive clonal lineage recombination leads to convergent evolution to a certain extent. Two grams Long lineage recombination rate is 10 times, and with different restriction modification system. For the first time in clinical isolates detected with the original receptor bacteria scattered in the sequence (interspersed sequences of the recipient, ISR) of the recombinant fragments, and found frequent recombination events caused by phage infection, suggesting that phages play an important role in the evolution of HP. The comparison between genomes showed that two clones had complete lineage while Cag pathogenicity island and the same sequence of Vac A, but the two clones are 50 KB difference, the complete encoding type 2 transposon TnPZ, for the further development of the type 2 TnPZ and HP pathogenic relevance, especially provides an important study on the relationship between patients with nephritis and clues. The second part: the expression of related gene of Helicobacter pylori resistant to amoxicillin and sequence analysis of HP AMO resistance divided into genetic resistance phenotype of resistance unstable and stable Two forms. This research chooses AMO HP 26695 as the starting strain sensitive strain induced by different concentrations of AMO AMO laboratory analysis of resistance HP., penicillin binding protein 5 (PBP1-PBP5), 2 holes (HopB and HopC) protein and an efflux pump protein (HefA) expression of.QRT-PCR encoding gene sequence variation results and the level of mRNA showed that the pressure in different concentration of AMO, HP detection of mRNA gene expression level is not the same.AMO below 4 mg/1 (0.5,1.0,2.0 mg/1), the expression level of PBP1 and PBP2 mRNA were higher than that of sensitive strains were more than 2 times, efflux pump protein HefA mRNA expression in AMO 0.5 and mg/l 1 concentration it was more than 2 times increase; at 4 mg/l under the pressure of AMO, mainly from PBP1 to PBP3 the expression of mRNA, HopB and HopC pore protein down regulated expression of mRNA and AMO reached 8; the concentration of mg/l, HopC and mRNA were down regulated. These results suggest that HP By increasing the expression of PBP, promote the synthesis of cell wall, increased efflux pump protein expression, increased drug efflux or reduce the pore proteins, reduce drug intake and other mechanisms to improve AMO resistance level. This study also obtained AMO genetic resistant strains, and PBP1 gene encoding newly discovered mutation sites related to T593K two 594G+ and AMO resistance. The study also found that the AMO pressure alone, can also cause varying degrees of resistance to levofloxacin, moxifloxacin resistance and high level tetracycline. The above results show that although the AMO resistance rate is low, stable resistant strains isolated from clinical rare, but AMO phenotypic resistance exists and because multiple use of AMO resulted in the occurrence of drug resistance, may be a factor in the declining rate of HP eradication can not be ignored. This phenomenon has not been reported. This study by the host HP and micro flora characteristics in Chemical analysis and the mechanism of phenotypic resistance and genetic resistance of HP AMO have deepened the understanding of HP evolution and genetic diversity, and the mechanism of AMO resistance, which has a certain significance for the follow-up HP pathogenic correlation research and HP prevention and treatment.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R692.3;R446.5

【共引文獻(xiàn)】

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本文編號(hào)：1722942