几种常用实验动物与人肠道主要菌群多样性比较

发布时间：2018-01-04 22:31

本文关键词：几种常用实验动物与人肠道主要菌群多样性比较　出处：《西南大学》2011年硕士论文　论文类型：学位论文

更多相关文章： 实验动物 肠道菌群 多样性 PCR-DGGE

【摘要】：人类是由10%的人体细胞和90%的微生物细胞共同组成的“超级生物体”。人的肠道中栖息着大约1014个,1000多种微生物,是人和动物体最庞大而复杂的生物群落,其主要分为与宿主共生的生理性细菌(类杆菌、双歧杆菌属、拟杆菌等),与宿主共栖的条件性致病菌(肠肝菌、肠球菌等)以及大多数为过路菌的病原菌(变形杆菌、霍乱弧菌、痢疾杆菌等),主要分布在结肠部位,正常情况下肠道菌群保持着共生和拮抗关系,从消化、营养吸收、能量供应、脂肪代谢、免疫调节、药物代谢和毒性等诸多方面影响人和动物的健康状况。对于肠道微生态的研究,传统的方法是通过微生物的选择性培养,或者是通过直接形态学观察来获得部分信息,再进行鉴定和分类。传统培养方法的局限性在于培养过程费时费力,易受操作方法的影响,敏感度低,大多数微生物很难或不能用现有的技术分离培养；培养技术只能定性检测可培养的细菌,不能鉴定未知的细菌,不能正确的反映肠道微生物群体的数量和多样性,使人类不能全面了解肠道微生物之间和与宿主的相互关系,阻碍了人类对肠道微生物的认识。近年来,随着分子生物学技术的迅猛发展,分子生物学技术在微生态学中的应用也日益广泛,其特点是能够快速获得微生物种群定性、定量数据,这使得微生态学现有的研究范围得以进一步扩展。以16S rRNA基因为基础的变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)技术能够快速准确地鉴定在自然环境或人工环境中的微生物种群,并进行复杂微生物群结构演替规律研究,以及生物种群的动态分析6。特别是近几年来国内外学者采用不同的分子生物学方法对细菌的16S rRNA进行研究,已经得到了广泛的16S rRNA序列数据库,为我们进行该比较微生态的研究提供了基础。目前用于肠道微生态研究的实验动物主要是用大/小鼠来建立的,其优点是个体小、易于操作、繁殖速度快、价格便宜等等,但是啮齿类动物在解剖、生理和代谢等方面与人之间存在较大的差异,在肠道微生态研究上并不能很好的作为模型动物,而就生理学、解剖学和营养代谢等方面小型猪与人更为相似,并且人的一些疾病如肝硬化,糖尿病、高血压和一些营养代谢症等在其身上也有发生,目前以小型猪作为实验材料的文章也越来越多。本研究利用PCR-DGGE技术对常用实验动物：FVB/n小鼠、BALB/c小鼠、SD (Sprague-Dawley)大鼠、Wister大鼠、巴马香猪、贵州小型猪与人的肠道总菌群、乳杆菌属菌群、拟杆菌属菌群进行了肠道菌群的多样性、丰富度和均匀度以及UPGMA相似性聚类分析。研究目的和意义通过比较微生态的方法,间接和直接阐明几种常用实验小鼠、大鼠、小型猪与人的肠道主要菌群在多样性的差异,正确反映它们作为常用模型动物与人之间肠道菌群的差异和相似性,初步分析上述几种常用医学实验动物肠道菌群的特点和差异,期望为肠道微生态研究提供基础性资料,以及在选择肠道微生态研究用模型动物时提供参考依据。研究方法 1.收集健康成年小鼠(FVB/n和BALB/c)、大鼠(SD和Wister)、小型猪(巴马香猪和贵州小型猪)和人的粪便样品,提取总菌DNA； 2.分别用V3(总菌)引物,lac(乳杆菌属)、bfr(拟杆菌属)二种特异性引物引物扩增提取到的肠道菌群总DNA、乳杆菌属菌群DNA和拟杆菌属菌群DNA； 3.分组进行变性梯度凝胶电泳(DGGE); 4.对每张电泳胶上的条带,利用spss13.0分析软件进行数据分析。研究结果利用PCR-DEEG方法,对两个品系小鼠,两种大鼠,两种小型猪,以及小鼠、大鼠、小型猪与人之间肠道总菌群、乳杆菌属和拟杆菌属的多样性、丰富度和均匀度分析,结果显示各种属内没有显著性差异,在种属间存在显著性差异,从肠道菌群同源性分析,小鼠、大鼠、小型猪的肠道总菌群、乳杆菌属菌群与人的比较,小型猪肠道菌群与人的最为接近,拟杆菌属比较实验动物之间比较接近。
[Abstract]:The human is composed of microbial cells of human cells 10% and 90% of the "super organism". The intestinal habitat of about 1014, 1000 kinds of microorganisms, biological communities in animal and human body the most large and complex, which is mainly divided into physiological and symbiotic bacterial host (Bacteroides, Bifidobacterium genus Bacteroides), and conditional pathogenic bacteria (host commensal intestinal bacteria Enterococcus and liver, etc.) for most passing bacteria pathogens (Vibrio cholerae, proteus, Shigella, etc.) are mainly distributed in the colon, normally the intestinal flora maintained a symbiotic and antagonistic relationship, from digestion. The absorption of nutrients, energy supply, fat metabolism, immune regulation, drug metabolism and toxicity in many aspects such as the influence of human and animal health.
For the study of intestinal microflora, the traditional method is through microbial selective culture, or to obtain information through direct observation of morphology, then the identification and classification of traditional culture. The limitation of the method lies in the training process is time consuming, easily affected by the method of operation, low sensitivity, most microorganisms are difficult or impossible to cultivate with the technology of the existing separation technology training; only qualitative detection of bacteria, identification of unknown bacteria can not reflect the number of intestinal microbial population, diversity and right can not fully understand the relationship between the human intestinal microflora between and with the host, hindering the understanding of human intestinal microflora. In recent years, with the rapid the development of molecular biology technology, the application of molecular biology technology in micro ecology is increasingly widespread, which is able to quickly obtain. Groups of species qualitative and quantitative data, which makes the research scope to further expand the existing micro ecology. Denaturing gradient gel electrophoresis with 16S based rRNA (denaturing gradient gel electrophoresis, DGGE) technology can rapidly and accurately identify the microbial population in the natural environment and artificial environment, and study the succession of microbial community structure complex well, the dynamic analysis of 6. biological populations, especially in recent years the domestic and foreign scholars using molecular biology methods on bacterial 16S rRNA research, has been 16S rRNA sequence database widely, the micro ecological research provides the basis for us.
Currently used experimental animal intestinal micro ecology research is mainly to build the large / mouse, the utility model has the advantages of small size, easy operation, fast propagation speed, low price and so on, but in the rodent animal anatomy, there is a big difference between physiology and metabolism and, in the study of intestinal flora and not as a good animal model, and physiology, anatomy and metabolism of small pig and human is more similar to human diabetes and some diseases such as cirrhosis, hypertension, and some nutritional metabolic disease has happened in the present, the pig as the experimental materials in more and more. This study uses PCR-DGGE technology to commonly used experimental animal: FVB/n mice, BALB/c mice, SD (Sprague-Dawley) rats, Wister rats, intestinal microflora of Bama miniature pig, Guizhou miniature pig and human, bacteria belonging to Lactobacillus, Bacteroides The diversity, richness and evenness of the intestinal flora, and the cluster analysis of UPGMA similarity were carried out in the bacterial flora.
The purpose and significance of the study
By comparing the micro ecological method, indirect and direct to clarify several commonly used experimental mice, rats, difference of intestinal microflora of pig and human in diversity, as they reflect the intestinal flora difference between human and animal model and similarity, a preliminary analysis of the characteristics and differences of the several common medical experiments animal intestinal flora, expect to provide basic data for the study of intestinal flora, as well as in the choice of intestinal microflora of animal models provide a reference.
research method
1. collect healthy adult mice (FVB/n and BALB/c), rats (SD and Wister), mini pigs (Bama miniature pig and Guizhou miniature pigs) and human fecal samples, extraction of total bacteria DNA;
2., the total DNA of intestinal flora, lactobacilli DNA and Bacteroides DNA were amplified by V3 (total bacterial) primers, Lac (Lactobacillus) and BFR (Bacteroides) two primers.
3. groups were divided into denaturing gradient gel electrophoresis (DGGE).
4. on the strip on each gel, SPSS13.0 analysis software is used to analyze the data.
Research results
Using PCR-DEEG method, two strains of mice, two rats, two mini pigs, rats, and mice, between miniature pig and human intestinal microflora, Lactobacillus and diversity of Bacteroides, richness and uniformity analysis, results showed no significant differences between the various species. In which there are significant differences between genera from the intestinal flora, homology analysis, mice, rats, intestinal microflora in pigs, Lactobacillus bacteria compared with people, small intestinal microflora and the most similar to Bacteroides compared relatively close to the animal.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378;S852.6

【参考文献】