基于高通量测序技术的枯草芽孢杆菌在体动态变化研究

发布时间：2017-12-26 15:55

本文关键词：基于高通量测序技术的枯草芽孢杆菌在体动态变化研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景肠道菌群在肠稳态维持和肠易激综合征(IBS)等多种疾病的发病机制中起重要作用。研究发现腹泻性IBS患者短期应用益生菌治疗可以调节异常的小肠粘膜的屏障功能,改善患者的腹痛和腹胀症状,并降低患者的总IBS症状评分。然而益生菌摄入后如何在肠道内定植以及如何影响肠道微生态尚不清楚。本实验设计并构建了"示踪基因",并应用16SrDNA高通量测序技术定量分析益生菌和肠道菌群的动态变化。目前对肠道微生物群的研究多应用传统的分子生物学技术,如实时荧光定量PCR和变性梯度凝胶电泳DGGE,这些方法具有一定的局限性。而Illumina高通量测序技术能够测序肠道中所有微生物DNA序列,全面分析肠道微生物落的多样性和丰度,满足对肠道微生物群落的全面认识。本实验采用高通量测序方法探究益生菌枯草芽孢杆菌(Bacillus subtilis)在体内的动态变化及其对肠道微生物群丰度和多样性的影响,并将高通量测序技术与传统的生物学技术——实时荧光定量PCR进行比较,进一步证实该技术在定量示踪益生菌时的优越性。目的探讨枯草芽孢杆菌在肠道内的定植情况及其服用时间长短对小鼠肠道菌群的影响。方法1.构建示踪基因:定量探究益生菌摄入后在肠道中的动态变化。为将摄入的益生菌与肠道固有菌群区分,本实验中动物实验部分使用的B.subtilis扩增细菌16S rRNA 的 338-806 区域,上下游引物分别为 338F-5' ACTCCTACGGGAGGCAGCA,806R-5' GGACTACHVGGGTWTCTAAT。经此示踪基因标记的益生菌在16SrDNA测序过程中会得到独有的循环序列,其丰度代表摄入干预益生菌B.subtilis的量,以此定量示踪益生菌在体内的动态变化。2.动物实验:18只C57BL/6J小鼠随机分成三组(n=6),分别为对照组(C组),两天组(TW组)和十天组(TE组),C组从第0天到第9天以0.9%生理盐水2 ml每天灌胃。TW组在第0天至第1天以B.subtilis 109 CFU/ml菌体悬液2 ml每天灌胃,第2天至第9天则以0.9%生理盐水2 ml每天灌胃。TE组从第0天至第9天以B.subtilis 109CFU/ml菌体悬液2 ml每天灌胃。三组小鼠均分别在第0天、第4天、第8天、第12天、第16天和第20天收集粪便并保存于-80℃冰箱。提取DNA后通过Illumina Miseq高通量测序平台测序,分析肠道菌群中各种门、属细菌的相对丰度及其变化规律,同时分析测序结果中示踪基因的相对丰度及其变化规律,代表摄入后益生菌的数量动态变化,探究肠道微生态环境对益生菌的作用规律。同时进行16SrDNA荧光定量PCR,将高通量测序和定量PCR两种方法的结果进行比较。结果1.灌胃后TW组和TE组的芽孢杆菌属微生物相对丰度与灌胃前相比下降。2.各组内肠道菌群相对丰度随时间变化规律为:厚壁菌门和拟杆菌的变化规律不明显,在益生菌灌胃的过程中呈反复波动的趋势,在第20天时稳定在十天组和两天组较对照组的厚壁菌门相对丰度更高;随着时间的变化两天组和十天组内变形菌门的相对丰度较实验组更低;疣微菌门出现了显著的而有规律性的变化:在第4天时TW组内的疣微菌门相对丰度开始较C组更高,但较TE组更低,其后随着时间变化一直稳定这种趋势,在第16天时出现短暂的高于TE组,但在第20天时相对丰度回落。而TE组内的疣微菌门相对丰度始终高于C组和TW组。3.TE组在除了第0天的各个时间点上小鼠肠道菌群结构相似,而TW组在这各个时间点的肠道菌群结构有存在差异。在第20天时,TW组和TE组小鼠肠道菌群结构相似。结论枯草芽孢杆菌并非通过定植,而是通过与肠道菌群相互作用使其结构发生改变的方式发挥益生功效;此外,长期服用该益生菌对肠道菌群结构的改变速度快于短期服用。
[Abstract]:Background intestinal microflora plays an important role in the pathogenesis of intestinal homeostasis and irritable bowel syndrome (IBS). It is found that short-term probiotics therapy can regulate abnormal intestinal mucosal barrier function, improve abdominal pain and abdominal distension symptoms, and reduce the total IBS symptom score of diarrhea patients with IBS. However, it is not clear how probiotics can be planted in the intestinal tract and how to affect the intestinal microflora after the intake of probiotics. In this experiment, the "tracer gene" was designed and constructed, and the dynamic changes of probiotics and intestinal flora were quantitatively analyzed by 16SrDNA high throughput sequencing technology. At present, the traditional molecular biology technology, such as real-time fluorescence quantitative PCR and denaturing gradient gel electrophoresis (DGGE), have some limitations in the study of intestinal microflora. Illumina high throughput sequencing technology can sequence all the DNA sequences of intestinal microflora, analyze the diversity and abundance of intestinal microbes, and satisfy the overall understanding of intestinal microbial communities. This experiment using high-throughput sequencing method of Probiotic Bacillus subtilis (Bacillus subtilis) in vivo and its dynamic changes of the intestinal microbiota abundance and diversity effects, and high-throughput sequencing technology and traditional biological technology -- comparison of real-time fluorescence quantitative PCR, further confirmed the superiority of this technique in the quantitative tracer of probiotics. Objective to investigate the effect of the colonization of Bacillus subtilis in intestinal tract and the effect of the length of taking time on the intestinal flora of mice. Method 1. the tracer gene was constructed: the quantitative exploration of the dynamic changes in the intestinal tract after the intake of probiotics. In order to distinguish the intake of probiotics from the intestinal flora, the 338-806 region of B.subtilis 16S rRNA was amplified in animal experiments. The upstream and downstream primers were 338F-5'ACTCCTACGGGAGGCAGCA, 806R-5' GGACTACHVGGGTWTCTAAT. The tracer gene labeled probiotics will get a unique cycle sequence in the process of 16SrDNA sequencing, and its abundance represents the amount of intervention probiotics B.subtilis, which can quantitatively trace the dynamic changes of probiotics in vivo. 2. animal experiment: 18 C57BL/6J mice were randomly divided into three groups (n=6), the control group (group C), the two day group (group TW) and the ten day group (TE group), C group from zeroth days to ninth days, 0.9% normal saline 2 ml daily gavage. TW group in zeroth to first days to B.subtilis 109 CFU/ml cell suspension 2 ml per day by gavage, second days to ninth days in 0.9% saline 2 ml per day by gavage. Group TE was intragastric from zeroth to ninth days with B.subtilis 109CFU/ml suspension 2 ml every day. The three groups of mice collected feces at zeroth, fourth, eighth, twelfth, sixteenth and twentieth days, respectively, and kept in the refrigerator at -80 C. After extracting DNA by Illumina Miseq sequencing platform sequencing, analysis of intestinal flora in all kinds of doors, the relative abundance of bacteria and its variation, and analysis of relative abundance of tracer gene sequencing results and its variation, represents the number of dynamic changes of probiotics, explore the role of intestinal microecology of probiotics. At the same time, 16SrDNA fluorescence quantitative PCR was carried out, and the results of high throughput sequencing and quantitative PCR two methods were compared. Results 1. after 1. gastric perfusion, the relative abundance of Bacillus in group TE and group TW decreased compared with that before gavage. 2. groups of intestinal flora changes with the relative abundance of time variation of Firmicutes and Bacteroidetes were not obvious, repeated fluctuations in the process of probiotics gavage, on the twentieth day in the relatively stable Firmicutes ten days group and two days group than in the control group more abundance high; with relative abundance of time variation of two days group and ten days group within the Proteobacteria was lower in experimental group; verrucomicrobia appear significant and regular changes: on the fourth day in TW group verrucomicrobia started relative abundance than C group is higher than TE, but group is lower, with the change of time has been stable following this trend, in sixteenth days was higher than that in group TE short, but in twentieth days when the relative abundance of fall. The relative abundance of verruca microphylum in group TE was always higher than that in group C and TW. In group 3.TE, the structure of intestinal flora in mice was similar at all time points except zeroth days, but there were differences in the structure of intestinal flora in group TW at all time points. At twentieth days, the structure of intestinal microflora in group TW and TE mice was similar. Conclusion Bacillus subtilis is not through colonization, but plays a prebiotic role by changing its structure through interaction with intestinal flora. Besides, long-term use of probiotics can change the structure of intestinal flora faster than short-term.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R57

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