肠道菌群结构变化与结直肠癌相关性的研究
发布时间：2018-08-17 09:50
【摘要】：目的:通过研究正常人与结直肠癌患者肠道微生物的多样性,了解两类人群肠道优势菌群,推测可能与结直肠癌的发生发展相关的菌属;并从微生物的角度分析左、右半结肠癌的菌群结构,探讨左右半结肠癌可能存在的差异。方法:获取8位正常人,8位右半结肠癌和5位左半结肠癌患者粪便菌群DNA后,利用保守区设计通用引物515F-806R进行PCR扩增并纯化,构建文库;使用HiSeq上机测序后,对原始数据进行拼接、过滤,获得有效数据,基于有效数据进行OTUs( Operational taxonomic units )聚类和物种分类分析。根据OTUs聚类结果,对每个OTU的代表序列做物种注释,得到对应的物种信息和基于物种的丰度分布情况;同时对OTUs进行丰度、Alpha多样性计算、Venn图和花瓣图等分析,一方面,获得了样品内物种丰富度和均匀度信息、不同样品或分组间的共有和特有OTUs信息等;另一方面,对OTUs进行多序列比对并构建系统发生树,进一步比较不同样品和分组的群落结构差异,并通过PCoA和PCA、NMDS等降维图和样品聚类树进行展示。同时,为进一步挖掘分组样品间的群落结构差异,选用T-test以及MetaStat统计分析方法对分组样品的物种组成和群落结果进行差异显著性检验。结果:通过对比正常人组与结直肠癌组的Chao1、ACE、simpson和Shannon指数,发现与正常人相比,结直肠癌患者肠道菌群的多样性相对较低,但是其菌群丰度相对较高。正常人与结直肠癌患者肠道菌群在门、纲、目、科、属的优势菌群及其丰度存在差异,主要表现为:在门水平:Firmicutes、Bacteroidetes、Proteobacteria;纲水平:Bacteroidales、Clostridiales、Selenomonadales、Burkholderizles、Enterobacteriales;目水平:Clotridiales、Bacteroidales、Enterobacteriales;科水平 Bacteroidaceae、Lachnospiraceae、Ruminococcaceae、Porphyromonadaceae;属水平:Bacteroides、Megamonas、Faecalibacterium、Parasutterella、Parabacteroides、Lachnoclostridium、Escherichia-Shigella、Subdoligranulum。结合系统发育树分析,发现对比正常组,肿瘤组表现为某些菌群丰度增加,如拟杆菌属、梭杆菌属、埃希氏菌属、粪肠球菌、卟啉单胞菌属等;同时一些菌群的丰度减少,如乳酸杆菌、双歧杆菌、罗氏菌属等。结论:1.正常人肠道微生物多样性及群落复杂程度更高。2.正常人与结直肠癌患者的肠道菌群在门、纲、目、科、及属水平的丰度上存在着一定差异。一些菌属在肿瘤患者中丰度较高,可能在肿瘤的发展过程中起到了推动作用,如拟杆菌属、梭杆菌属、埃希氏菌属等;另一些菌属在肿瘤患者中丰度较低,他们对正常的机体可能有益处,如乳酸杆菌、双歧杆菌、罗氏菌属等。3.左右半结肠癌患者的肠道优势菌群菌群在门、纲、目、科、及属水平的丰度上存在着一定差异。右半结肠癌患者属水平的优势菌群为Fusobacteria,Bacteroidetes,Lachnospiraceae,Enterobacteriaceae;左半结肠癌患者属水平的优势菌群为 Bacteroidetes,B. Fragilis,Lachnospiraceae,Fusobacteria;肠道菌群在左、右半结肠癌肠道的生物学特性可能存在差异。
[Abstract]:Objective: To study the diversity of intestinal microorganisms in normal people and patients with colorectal cancer, to understand the dominant intestinal flora of the two groups of people, and to speculate the bacterial genus which may be related to the occurrence and development of colorectal cancer. The DNA of stool flora from 8 normal subjects, 8 right colon cancer patients and 5 left colon cancer patients was amplified and purified by PCR using a universal primer 515F-806R designed by conservative region, and the library was constructed. Clustering and species classification analysis. According to the OTUs clustering results, the representative sequences of each OTU are annotated to get the corresponding species information and the species abundance distribution based on the species. Meanwhile, the OTUs are analyzed by abundance, Alpha diversity calculation, Venn diagram and petal diagram. On the one hand, the species richness and evenness information in the sample is obtained. On the other hand, OTUs were sequentially aligned and phylogenetic trees were constructed to further compare the differences of community structure between different samples and groups, and then displayed by dimensionality reduction maps such as PCoA, PCA, NMDS and sample clustering tree. Results: By comparing the Chao1, ACE, Simpson and Shannon indices of normal and colorectal cancer patients, it was found that the diversity of intestinal flora in colorectal cancer patients was lower than that in normal subjects, but the diversity of intestinal flora was lower. The dominant intestinal flora and its abundance in the phylum, class, order, family and genus of normal people and patients with colorectal cancer are different, mainly as follows: at the phylum level: Firmicutes, Bacteroidetes, Proteobacteria; class level: Bacteroidales, Clostridiales, Selenomonadales, Burkholderizles, Enterobacteriales; class level: Firmicutes, Bacteroidetes, Bacteroidetes, Proteobacteria; class level: Bacteroidales, Clostridiales, Selenomonadales, Burkholderiz Tridiales, Bacteroidales, Enterobacteriales; Family level Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Porphyromonadaceae; Genus level: Bacteroides, Megamonas, Faecalibacterium, Parasutterella, Parabacteroides, Lachnoclostridium, Escherichia-Shigella, Doligranulum. In group B, the abundance of some bacteria increased, such as Bacteroides, Fusobacterium, Escherichia coli, Enterococcus faecalis, Porphyromonas, and so on. At the same time, the abundance of some bacteria decreased, such as Lactobacillus, Bifidobacterium, Rochella and so on. Conclusion: 1. The intestinal microbial diversity and community complexity of normal people and colorectal cancer were higher. There are certain differences in the abundance of intestinal flora among cancer patients at the phylum, class, order, family, and genus levels. Some bacteria are abundant in cancer patients and may play a role in promoting the development of cancer, such as Bacteroides, Fusobacterium, Escherichia, etc. Others are low in cancer patients and they are normal to them. There are some differences in the abundance of the dominant flora in the intestine between the right and left hemicolonal cancer patients at the phylum, class, order, family, and genus levels. The dominant flora in the right hemicolonal cancer patients at the genus level are Fusobacteria, Bacteroidetes, Lachnospiraceae, Enterobacteriaceae. Bacteroidetes, B. Fragilis, Lachnospiraceae, Fusobacteria were the predominant flora in the genus of hemicolonal cancer patients, and the biological characteristics of the left and right colon cancer may be different.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2187232
[Abstract]:Objective: To study the diversity of intestinal microorganisms in normal people and patients with colorectal cancer, to understand the dominant intestinal flora of the two groups of people, and to speculate the bacterial genus which may be related to the occurrence and development of colorectal cancer. The DNA of stool flora from 8 normal subjects, 8 right colon cancer patients and 5 left colon cancer patients was amplified and purified by PCR using a universal primer 515F-806R designed by conservative region, and the library was constructed. Clustering and species classification analysis. According to the OTUs clustering results, the representative sequences of each OTU are annotated to get the corresponding species information and the species abundance distribution based on the species. Meanwhile, the OTUs are analyzed by abundance, Alpha diversity calculation, Venn diagram and petal diagram. On the one hand, the species richness and evenness information in the sample is obtained. On the other hand, OTUs were sequentially aligned and phylogenetic trees were constructed to further compare the differences of community structure between different samples and groups, and then displayed by dimensionality reduction maps such as PCoA, PCA, NMDS and sample clustering tree. Results: By comparing the Chao1, ACE, Simpson and Shannon indices of normal and colorectal cancer patients, it was found that the diversity of intestinal flora in colorectal cancer patients was lower than that in normal subjects, but the diversity of intestinal flora was lower. The dominant intestinal flora and its abundance in the phylum, class, order, family and genus of normal people and patients with colorectal cancer are different, mainly as follows: at the phylum level: Firmicutes, Bacteroidetes, Proteobacteria; class level: Bacteroidales, Clostridiales, Selenomonadales, Burkholderizles, Enterobacteriales; class level: Firmicutes, Bacteroidetes, Bacteroidetes, Proteobacteria; class level: Bacteroidales, Clostridiales, Selenomonadales, Burkholderiz Tridiales, Bacteroidales, Enterobacteriales; Family level Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Porphyromonadaceae; Genus level: Bacteroides, Megamonas, Faecalibacterium, Parasutterella, Parabacteroides, Lachnoclostridium, Escherichia-Shigella, Doligranulum. In group B, the abundance of some bacteria increased, such as Bacteroides, Fusobacterium, Escherichia coli, Enterococcus faecalis, Porphyromonas, and so on. At the same time, the abundance of some bacteria decreased, such as Lactobacillus, Bifidobacterium, Rochella and so on. Conclusion: 1. The intestinal microbial diversity and community complexity of normal people and colorectal cancer were higher. There are certain differences in the abundance of intestinal flora among cancer patients at the phylum, class, order, family, and genus levels. Some bacteria are abundant in cancer patients and may play a role in promoting the development of cancer, such as Bacteroides, Fusobacterium, Escherichia, etc. Others are low in cancer patients and they are normal to them. There are some differences in the abundance of the dominant flora in the intestine between the right and left hemicolonal cancer patients at the phylum, class, order, family, and genus levels. The dominant flora in the right hemicolonal cancer patients at the genus level are Fusobacteria, Bacteroidetes, Lachnospiraceae, Enterobacteriaceae. Bacteroidetes, B. Fragilis, Lachnospiraceae, Fusobacteria were the predominant flora in the genus of hemicolonal cancer patients, and the biological characteristics of the left and right colon cancer may be different.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
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