本文選題：胃部菌群　切入點(diǎn)：16S　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:胃潰瘍(Gastric Ulcer,GU)是消化性潰瘍的一種,全世界約5%-10%的人都患有過胃潰瘍。胃作為消化系統(tǒng)中的重要組成部分,存在許多特殊的微生物群落組成的菌群結(jié)構(gòu)。胃部微生物群落結(jié)構(gòu)及其所執(zhí)行的功能,尤其是與人類胃部疾病關(guān)系尚不明確。已有的少量研究多集中在胃部菌群結(jié)構(gòu)與惡性胃部疾病(胃癌等)關(guān)系上,對(duì)胃潰瘍患者胃部菌群結(jié)構(gòu)的解讀少之又少。胃潰瘍患者胃部菌群結(jié)構(gòu)特點(diǎn)及其與健康人群之間的差異亟待闡明。故本研究從消化道微生態(tài)角度出發(fā),利用16S r DNA高通量測序技術(shù)和生物信息學(xué)方法,分析胃潰瘍患者與健康人群胃竇組織菌群結(jié)構(gòu)特點(diǎn)及差異,構(gòu)建胃潰瘍顯著特異性菌群構(gòu)成圖譜,并篩選疾病顯著相關(guān)的關(guān)鍵菌群或菌種,進(jìn)一步對(duì)從患者胃竇組織分離的幽門螺桿菌(Helicobacter pylori,H.pylori),進(jìn)行分子進(jìn)化研究,并結(jié)合患者胃部菌群結(jié)構(gòu)進(jìn)行關(guān)聯(lián)分析。本研究將為胃部菌群結(jié)構(gòu)與胃潰瘍發(fā)生發(fā)展關(guān)系及內(nèi)在機(jī)制的闡明打下基礎(chǔ),同時(shí)為胃潰瘍的臨床治療提供新的思路。方法:收集胃潰瘍患者和健康查體人員胃竇組織樣本,提取樣品的總DNA,PCR擴(kuò)增16S r DNA基因V3-V4可變區(qū),產(chǎn)物使用Illumina Mi Seq高通量測序儀測序。下機(jī)數(shù)據(jù)經(jīng)預(yù)處理獲得每個(gè)樣本的高質(zhì)量序列。進(jìn)一步進(jìn)行OTU劃分、共有OTU分析及分類地位鑒定考查樣本在各分類水平的OTU分布情況。通過繪制稀疏曲線、物種累積曲線、豐富等級(jí)曲線等評(píng)價(jià)菌群α多樣性。根據(jù)樣本在各分類學(xué)水平的組成和序列分布,比較每個(gè)分類單元在兩組之間的豐度差異,并以熱圖展示。通過主成分分析與非度量多維尺度分析,評(píng)價(jià)菌群結(jié)構(gòu)的β多樣性。根據(jù)優(yōu)勢屬在不同樣本中的豐度分布,建胃部優(yōu)勢微生物類群的關(guān)聯(lián)網(wǎng)絡(luò)。從胃潰瘍患者胃竇組織中分離培養(yǎng)H.pylori,PCR擴(kuò)增cag A基因的3’端可變區(qū)序列,產(chǎn)物測序后用于繪制系統(tǒng)發(fā)育樹。分析Cag A蛋白羧基端EPIYA位點(diǎn)特征并結(jié)合患者胃部菌群結(jié)構(gòu)進(jìn)行關(guān)聯(lián)分析。結(jié)果:本研究采集健康人胃竇組織樣本10例,胃潰瘍患者胃竇組織樣本9例。細(xì)菌16S r DNA的v3-v4區(qū)擴(kuò)增結(jié)果顯示,所有樣本在500bp左右有明顯條帶,所獲目的片段純度較高,滿足測序要求。對(duì)下機(jī)數(shù)據(jù)預(yù)處理后,OTU分析發(fā)現(xiàn),健康組在各分類水平上的OTU數(shù)均顯著高于胃潰瘍組,兩組之間共有OTU1172個(gè),健康組獨(dú)有OTU 413個(gè),胃潰瘍組92個(gè)。樣本間α多樣性評(píng)估證實(shí)測序量與測序深度足以覆蓋胃內(nèi)細(xì)菌種屬,兩組樣本含有的物種豐富程度和均勻程度良好。對(duì)比健康人和胃潰瘍患者胃部菌群α多樣性發(fā)現(xiàn),在抽取相同數(shù)目序列情況下,健康組觀察到的物種數(shù)、chao1指數(shù)和Shannon多樣性指數(shù)均高于疾病組。將OTU注釋到各分類水平,來自30個(gè)菌門的250多個(gè)菌屬被鑒定出來。所有胃竇粘膜樣本中優(yōu)勢菌門均依次為:變形菌門、厚壁菌門、放線菌門和擬桿菌門。胃潰瘍組和健康組相比豐度有顯著差異的細(xì)菌門類依次為:擬桿菌門、厚壁菌門、變形菌門、酸桿菌門、梭桿菌門、放線菌門、軟壁菌門、綠彎菌門、互養(yǎng)菌門。在屬水平上,胃潰瘍組螺桿菌屬的豐度明顯高于健康組,而在乳球菌屬、擬桿菌屬、放線菌屬等19個(gè)屬上,豐度顯著低于健康組。菌群結(jié)構(gòu)的Beta多樣性分析發(fā)現(xiàn),健康組和胃潰瘍組的組間微生物群落結(jié)構(gòu)差異明顯,相似度較低。網(wǎng)絡(luò)分析顯示,胃內(nèi)優(yōu)勢菌屬主要分為兩個(gè)典型集團(tuán),兩集團(tuán)間樞紐為螺桿菌屬。從胃潰瘍患者胃竇組織中分離鑒定得到9株H.pylori,分離率100%。cag A基因擴(kuò)增結(jié)果顯示,9株菌在500-800bp左右均有明顯條帶。分子進(jìn)化分析發(fā)現(xiàn),9株菌均為東亞型。Cag A蛋白羧基端多態(tài)性位點(diǎn)分析顯示,JLU-1和JLU-9為EPIYA-ABDD型,JLU-4為EPIYA-AB型,其余均為EPIYA-ABD型。結(jié)合患者胃部菌群結(jié)構(gòu)進(jìn)行關(guān)聯(lián)分析,結(jié)果顯示EPIYA-D位點(diǎn)的數(shù)目與患者胃部H.pylori的相對(duì)豐度呈正相關(guān)。結(jié)論:1.本研究應(yīng)用細(xì)菌16S r DNA高通量測序技術(shù),對(duì)9例胃潰瘍患者和10例健康人進(jìn)行胃部菌群分布分析發(fā)現(xiàn):胃潰瘍患者和健康人胃部均存在大量變形菌門、擬桿菌門、放線菌門和厚壁菌門細(xì)菌;胃潰瘍患者的胃部菌群,在門水平上以變形菌門為主(占80%),屬水平上以螺桿菌屬為主(占50%);與健康人相比,胃潰瘍患者胃部菌群分布均勻度差、多樣性明顯降低,在變形菌門、擬桿菌門、放線菌門等9個(gè)門及螺桿菌屬、乳球菌屬、放線菌屬等20個(gè)屬的豐度上具有顯著差異。2.本研究從9例長春地區(qū)胃潰瘍患者胃竇組織中均分離得到H.pylori,基于cag A基因繪制系統(tǒng)進(jìn)化樹,發(fā)現(xiàn)分離的H.pylori的cag A均為東亞型。3.首先發(fā)現(xiàn)長春地區(qū)胃潰瘍患者H.pylori臨床分離株Cag A蛋白羧基端EPIYA-D位點(diǎn)的重復(fù)性可能與H.pylori在患者胃部的定植有關(guān)。
[Abstract]:Background: gastric ulcer (Gastric Ulcer GU) is a kind of peptic ulcer, the whole world is about 5%-10% people suffering from stomach ulcers. As an important part of the digestive system, there are many special flora composition of the microbial community. The stomach microbial community structure and functions especially in relation to human stomach disease is not clear. The few researches focus on the stomach flora and malignant gastric diseases (cancer) relationship, analysis of patients with gastric ulcer gastric microflora. There is little difference between the gastric ulcer patients, flora characteristics and healthy people to clarify this research. From the digestive tract micro ecology perspective, the use of 16S R DNA high-throughput sequencing and bioinformatics methods, analysis of patients with gastric ulcer and gastric antrum healthy flora structure characteristics and difference of construction Gastric ulcer was specific bacterial composition profiles, and screening significant diseases related to key bacteria or fungi, further on Helicobacter pylori isolated from patients with gastric antral tissue (Helicobacter, pylori, H.pylori) for the study of molecular evolution, and combined with the stomach flora in patients with structure of correlation analysis. This study will be related to the development and the internal mechanism for stomach microflora and gastric ulcer that lay the foundation at the same time, to provide new ideas for clinical treatment of gastric ulcer. Methods: We collected gastric ulcer patients and healthy persons gastric tissue samples, DNA extraction sample, PCR amplification of 16S R gene DNA V3-V4 variable region, the use of Illumina products Mi Seq high-throughput sequencing machine. Under the data after preprocessing to obtain high quality sequences of each sample. Further to OTU division, total and taxonomic status analysis and identification of OTU samples in each level of O The distribution of TU. By drawing the sparse curve, species accumulation curve, rich rating curve flora diversity. According to the composition and sequence distribution of samples in each taxonomic level, comparative abundance differences between the two groups in each classification unit, and image display. Through principal component analysis and non metric multidimensional scaling. Evaluation of microbial community structure diversity. According to the abundance distribution of dominant species in different samples, associated network microorganism stomach advantage. H.pylori were isolated and cultured from patients with gastric antrum gastric ulcer, PCR amplification of CAG A gene 3 'end sequences of variable region, sequencing for phylogenetic tree analysis. Cag A protein C-terminal EPIYA sites and combined with the features of stomach flora in patients with structure of correlation analysis. Results: This study collected healthy gastric tissue samples from 10 cases of gastric ulcer patients, 9 cases of gastric antrum tissue samples. R DNA v3-v4 bacterial 16S amplification results showed that all the samples have obvious bands at about 500bp, the fragment of high purity, to meet the requirements of machine sequencing. After data preprocessing, OTU analysis found that the health group number OTU in each classification level were significantly higher than those in gastric ulcer group, between the two groups there are OTU1172, OTU 413 unique health group, gastric ulcer group 92. Sample diversity assessment proved order quantity and sequencing depth enough to cover the stomach bacteria species, two groups of samples containing species richness and uniformity is good. Compared with healthy people and patients with gastric ulcer stomach bacteria alpha diversity that, in the same number of sequence extraction cases, health groups observed species richness, Chao1 index and Shannon diversity index were higher than the disease group. OTU notes to each classification level, from 30 of the more than 250 bacterial genera were identified. All the gastric antrum 綺樿啘鏍鋒湰涓紭鍔胯弻闂ㄥ潎渚濇涓，

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