胃溃疡患者胃部菌群结构分析及幽门螺杆菌分子进化研究

发布时间：2018-03-16 01:38

  本文选题：胃部菌群　切入点：16S　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:胃溃疡(Gastric Ulcer,GU)是消化性溃疡的一种,全世界约5%-10%的人都患有过胃溃疡。胃作为消化系统中的重要组成部分,存在许多特殊的微生物群落组成的菌群结构。胃部微生物群落结构及其所执行的功能,尤其是与人类胃部疾病关系尚不明确。已有的少量研究多集中在胃部菌群结构与恶性胃部疾病(胃癌等)关系上,对胃溃疡患者胃部菌群结构的解读少之又少。胃溃疡患者胃部菌群结构特点及其与健康人群之间的差异亟待阐明。故本研究从消化道微生态角度出发,利用16S r DNA高通量测序技术和生物信息学方法,分析胃溃疡患者与健康人群胃窦组织菌群结构特点及差异,构建胃溃疡显著特异性菌群构成图谱,并筛选疾病显著相关的关键菌群或菌种,进一步对从患者胃窦组织分离的幽门螺杆菌(Helicobacter pylori,H.pylori),进行分子进化研究,并结合患者胃部菌群结构进行关联分析。本研究将为胃部菌群结构与胃溃疡发生发展关系及内在机制的阐明打下基础,同时为胃溃疡的临床治疗提供新的思路。方法:收集胃溃疡患者和健康查体人员胃窦组织样本,提取样品的总DNA,PCR扩增16S r DNA基因V3-V4可变区,产物使用Illumina Mi Seq高通量测序仪测序。下机数据经预处理获得每个样本的高质量序列。进一步进行OTU划分、共有OTU分析及分类地位鉴定考查样本在各分类水平的OTU分布情况。通过绘制稀疏曲线、物种累积曲线、丰富等级曲线等评价菌群α多样性。根据样本在各分类学水平的组成和序列分布,比较每个分类单元在两组之间的丰度差异,并以热图展示。通过主成分分析与非度量多维尺度分析,评价菌群结构的β多样性。根据优势属在不同样本中的丰度分布,建胃部优势微生物类群的关联网络。从胃溃疡患者胃窦组织中分离培养H.pylori,PCR扩增cag A基因的3’端可变区序列,产物测序后用于绘制系统发育树。分析Cag A蛋白羧基端EPIYA位点特征并结合患者胃部菌群结构进行关联分析。结果:本研究采集健康人胃窦组织样本10例,胃溃疡患者胃窦组织样本9例。细菌16S r DNA的v3-v4区扩增结果显示,所有样本在500bp左右有明显条带,所获目的片段纯度较高,满足测序要求。对下机数据预处理后,OTU分析发现,健康组在各分类水平上的OTU数均显著高于胃溃疡组,两组之间共有OTU1172个,健康组独有OTU 413个,胃溃疡组92个。样本间α多样性评估证实测序量与测序深度足以覆盖胃内细菌种属,两组样本含有的物种丰富程度和均匀程度良好。对比健康人和胃溃疡患者胃部菌群α多样性发现,在抽取相同数目序列情况下,健康组观察到的物种数、chao1指数和Shannon多样性指数均高于疾病组。将OTU注释到各分类水平,来自30个菌门的250多个菌属被鉴定出来。所有胃窦粘膜样本中优势菌门均依次为:变形菌门、厚壁菌门、放线菌门和拟杆菌门。胃溃疡组和健康组相比丰度有显著差异的细菌门类依次为:拟杆菌门、厚壁菌门、变形菌门、酸杆菌门、梭杆菌门、放线菌门、软壁菌门、绿弯菌门、互养菌门。在属水平上,胃溃疡组螺杆菌属的丰度明显高于健康组,而在乳球菌属、拟杆菌属、放线菌属等19个属上,丰度显著低于健康组。菌群结构的Beta多样性分析发现,健康组和胃溃疡组的组间微生物群落结构差异明显,相似度较低。网络分析显示,胃内优势菌属主要分为两个典型集团,两集团间枢纽为螺杆菌属。从胃溃疡患者胃窦组织中分离鉴定得到9株H.pylori,分离率100%。cag A基因扩增结果显示,9株菌在500-800bp左右均有明显条带。分子进化分析发现,9株菌均为东亚型。Cag A蛋白羧基端多态性位点分析显示,JLU-1和JLU-9为EPIYA-ABDD型,JLU-4为EPIYA-AB型,其余均为EPIYA-ABD型。结合患者胃部菌群结构进行关联分析,结果显示EPIYA-D位点的数目与患者胃部H.pylori的相对丰度呈正相关。结论:1.本研究应用细菌16S r DNA高通量测序技术,对9例胃溃疡患者和10例健康人进行胃部菌群分布分析发现:胃溃疡患者和健康人胃部均存在大量变形菌门、拟杆菌门、放线菌门和厚壁菌门细菌;胃溃疡患者的胃部菌群,在门水平上以变形菌门为主(占80%),属水平上以螺杆菌属为主(占50%);与健康人相比,胃溃疡患者胃部菌群分布均匀度差、多样性明显降低,在变形菌门、拟杆菌门、放线菌门等9个门及螺杆菌属、乳球菌属、放线菌属等20个属的丰度上具有显著差异。2.本研究从9例长春地区胃溃疡患者胃窦组织中均分离得到H.pylori,基于cag A基因绘制系统进化树,发现分离的H.pylori的cag A均为东亚型。3.首先发现长春地区胃溃疡患者H.pylori临床分离株Cag A蛋白羧基端EPIYA-D位点的重复性可能与H.pylori在患者胃部的定植有关。
[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 绮樿啘鏍锋湰涓�浼樺娍鑿岄棬鍧囦緷娆′福�

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