本文關(guān)鍵詞：幾種常用實驗動物與人腸道主要菌群多樣性比較　出處：《西南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 實驗動物 腸道菌群 多樣性 PCR-DGGE

【摘要】：人類是由10%的人體細(xì)胞和90%的微生物細(xì)胞共同組成的“超級生物體”。人的腸道中棲息著大約1014個,1000多種微生物,是人和動物體最龐大而復(fù)雜的生物群落,其主要分為與宿主共生的生理性細(xì)菌(類桿菌、雙歧桿菌屬、擬桿菌等),與宿主共棲的條件性致病菌(腸肝菌、腸球菌等)以及大多數(shù)為過路菌的病原菌(變形桿菌、霍亂弧菌、痢疾桿菌等),主要分布在結(jié)腸部位,正常情況下腸道菌群保持著共生和拮抗關(guān)系,從消化、營養(yǎng)吸收、能量供應(yīng)、脂肪代謝、免疫調(diào)節(jié)、藥物代謝和毒性等諸多方面影響人和動物的健康狀況。 對于腸道微生態(tài)的研究,傳統(tǒng)的方法是通過微生物的選擇性培養(yǎng),或者是通過直接形態(tài)學(xué)觀察來獲得部分信息,再進(jìn)行鑒定和分類。傳統(tǒng)培養(yǎng)方法的局限性在于培養(yǎng)過程費時費力,易受操作方法的影響,敏感度低,大多數(shù)微生物很難或不能用現(xiàn)有的技術(shù)分離培養(yǎng)；培養(yǎng)技術(shù)只能定性檢測可培養(yǎng)的細(xì)菌,不能鑒定未知的細(xì)菌,不能正確的反映腸道微生物群體的數(shù)量和多樣性,使人類不能全面了解腸道微生物之間和與宿主的相互關(guān)系,阻礙了人類對腸道微生物的認(rèn)識。近年來,隨著分子生物學(xué)技術(shù)的迅猛發(fā)展,分子生物學(xué)技術(shù)在微生態(tài)學(xué)中的應(yīng)用也日益廣泛,其特點是能夠快速獲得微生物種群定性、定量數(shù)據(jù),這使得微生態(tài)學(xué)現(xiàn)有的研究范圍得以進(jìn)一步擴展。以16S rRNA基因為基礎(chǔ)的變性梯度凝膠電泳(denaturing gradient gel electrophoresis, DGGE)技術(shù)能夠快速準(zhǔn)確地鑒定在自然環(huán)境或人工環(huán)境中的微生物種群,并進(jìn)行復(fù)雜微生物群結(jié)構(gòu)演替規(guī)律研究,以及生物種群的動態(tài)分析6。特別是近幾年來國內(nèi)外學(xué)者采用不同的分子生物學(xué)方法對細(xì)菌的16S rRNA進(jìn)行研究,已經(jīng)得到了廣泛的16S rRNA序列數(shù)據(jù)庫,為我們進(jìn)行該比較微生態(tài)的研究提供了基礎(chǔ)。 目前用于腸道微生態(tài)研究的實驗動物主要是用大/小鼠來建立的,其優(yōu)點是個體小、易于操作、繁殖速度快、價格便宜等等,但是嚙齒類動物在解剖、生理和代謝等方面與人之間存在較大的差異,在腸道微生態(tài)研究上并不能很好的作為模型動物,而就生理學(xué)、解剖學(xué)和營養(yǎng)代謝等方面小型豬與人更為相似,并且人的一些疾病如肝硬化,糖尿病、高血壓和一些營養(yǎng)代謝癥等在其身上也有發(fā)生,目前以小型豬作為實驗材料的文章也越來越多。本研究利用PCR-DGGE技術(shù)對常用實驗動物：FVB/n小鼠、BALB/c小鼠、SD (Sprague-Dawley)大鼠、Wister大鼠、巴馬香豬、貴州小型豬與人的腸道總菌群、乳桿菌屬菌群、擬桿菌屬菌群進(jìn)行了腸道菌群的多樣性、豐富度和均勻度以及UPGMA相似性聚類分析。 研究目的和意義 通過比較微生態(tài)的方法,間接和直接闡明幾種常用實驗小鼠、大鼠、小型豬與人的腸道主要菌群在多樣性的差異,正確反映它們作為常用模型動物與人之間腸道菌群的差異和相似性,初步分析上述幾種常用醫(yī)學(xué)實驗動物腸道菌群的特點和差異,期望為腸道微生態(tài)研究提供基礎(chǔ)性資料,以及在選擇腸道微生態(tài)研究用模型動物時提供參考依據(jù)。 研究方法 1.收集健康成年小鼠(FVB/n和BALB/c)、大鼠(SD和Wister)、小型豬(巴馬香豬和貴州小型豬)和人的糞便樣品,提取總菌DNA； 2.分別用V3(總菌)引物,lac(乳桿菌屬)、bfr(擬桿菌屬)二種特異性引物引物擴增提取到的腸道菌群總DNA、乳桿菌屬菌群DNA和擬桿菌屬菌群DNA； 3.分組進(jìn)行變性梯度凝膠電泳(DGGE); 4.對每張電泳膠上的條帶,利用spss13.0分析軟件進(jìn)行數(shù)據(jù)分析。 研究結(jié)果 利用PCR-DEEG方法,對兩個品系小鼠,兩種大鼠,兩種小型豬,以及小鼠、大鼠、小型豬與人之間腸道總菌群、乳桿菌屬和擬桿菌屬的多樣性、豐富度和均勻度分析,結(jié)果顯示各種屬內(nèi)沒有顯著性差異,在種屬間存在顯著性差異,從腸道菌群同源性分析,小鼠、大鼠、小型豬的腸道總菌群、乳桿菌屬菌群與人的比較,小型豬腸道菌群與人的最為接近,擬桿菌屬比較實驗動物之間比較接近。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378;S852.6

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