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1、上海交通大学 硕士学位论文 1型糖尿病鼠类模型的肠道菌群分析 姓名：徐灵筠 申请学位级别：硕士 专业：微生物分子生态学与基因组学 指导教师：赵立平 20070101 上海交通大学理学硕士学位论文 ABSTRACT 1 型糖尿病鼠类模型的肠道菌群分析 摘 要 1 型糖尿病鼠类模型的肠道菌群分析 摘 要 近年来，1 型糖尿病及其动物模型的发病机理已经研究得较为透彻， 鼠类模型的发病过程被归纳为遗传缺陷主导，环境因素诱发，两者相互 作用导致了 1 型糖尿病的发生。环境因素的研究主要集中于营养物和病 毒感染，肠道微生态系统因其内在复杂性而少有涉及。少数相关的文献 用分离培养的方法揭示了糖尿病病人和模型
2、动物发生了肠道菌群失调。 我们实验室采用分子生态学的方法来研究两者的关系，发现肠道菌群与 糖尿病的发生发展存在一定的关系，某些菌可能与糖尿病直接相关。 实验中，我们将同一批 ICR 小鼠，随机分成两组，一组为糖尿病模 型组，另外一组为健康对照组。糖尿病造模采用静脉注射链脲佐菌素 （STZ）方法，造模后糖尿病小鼠体重明显轻于对照小鼠，表现出显著糖 尿病“三多一少”的症状。保留几只血糖未升高的造模鼠，与造模成功 小鼠的作对照。 首先采用 ERIC-PCR 指纹图谱技术解析各组小鼠的粪便菌群总 DNA 样品，获得 DNA 指纹信息，通过 PLS-DA（偏最小二乘法区别分析）统计 方法寻找各组样品之间
3、的规律性变化，结果发现，对 STZ 敏感的糖尿病 小鼠和对 STZ 不敏感的小鼠的肠道菌群显著区别于健康对照小鼠的菌 群，而 STZ 易感性小鼠与 STZ 抵抗型小鼠的菌群仍有一定差别。为了进 一步辨别菌群结构的差异和决定此差异的菌种，我们又采用了 16S rDNA V3DGGE 方法对样品进行了分析，结果显示，所有空白对照与造模不 上海交通大学理学硕士学位论文 ABSTRACT 成功的鼠（低血糖）都有两条较亮的条带，而糖尿病小鼠则较暗。割胶 回收 DNA，测序结果表明这两种都属于乳杆菌属（Lactobacillus） 。因此我 们选用乳酸菌种群（LAB）特异性引物扩增所有样品，DGGE 的分
4、析结果 验证了低血糖组的两条明显的优势带在高血糖组中被显著减弱。经测序 分析，这两条带分别代表肠乳酸杆菌（Lactobacillus intestinalis）和罗伊 氏乳酸杆菌(Lactobacillus reuteri)。最后，通过 V3 DGGE 和 LAB DGGE 图 谱的主成分分析分析，对比三组小鼠菌群的条带变化，验证了这两种乳 杆菌是导致低血糖和高血糖两类小鼠肠道菌群差别的主要原因，而其他 健康相关菌群拟杆菌、梭菌、双歧杆菌的群落结构未见明显差别。 此外，在糖尿病小鼠的试验中，还观察到糖尿病小鼠的双歧杆菌数量明 显低于正常小鼠。以上结果表明糖尿病发生后，两种与宿主健康密切相 关的
5、肠道细菌群落（双歧杆菌和乳杆菌）分别发生了结构上和数量上的 改变。 此后,对糖尿病大鼠的肠道菌群结构的初步分析,表明糖尿病大鼠的 乳酸菌种群结构随时间的变化显著与健康对照大鼠的不同，而两者的菌 群结构在造模前时间点上没有明显差异。这提示糖尿病的发生和发展是 导致肠道菌群结构发生变化的重要影响因素。 本研究中，肠道益生菌的结构数量变化与血糖值的反相关对应关系， 与采用分离培养的肠道菌群研究所揭示的结果相类似。这表明糖尿病鼠 类发生的肠道菌群失调主要表现为肠道益生菌的减少，因此可能会影响 宿主糖尿病的发生和发展，补充益生菌或用药物干预菌群结构可能将成 为预防和治疗 1 型糖尿病的有效途径。 关键词
6、：关键词：肠道菌群，1 型糖尿病，链脲佐菌素（STZ） ，乳杆菌，双歧杆 菌，益生菌，ERIC-PCR，PCR-DGGE，PCA，PLS-DA 上海交通大学理学硕士学位论文 ABSTRACT Intestinal Microflora Analysis for Type 1 Diabetic Murine ABSTRACT In recent years, the pathology of type 1 diabetes mellitus and its animal model is nearly well studied. The pathogenesis of diabetic muri
7、ne model is thought to be determined by genetic defects, triggered by environment factors. Their close interaction results in the onset of type 1 diabetes mellitus. The former research on environmental factors mainly focuses on nutrient and virus infection, while little is touched on gut microecolog
8、ical system probably because of its inner complexity. Literature disclosed the disorder of intestinal microflora occurs in diabetic patients and animal models through isolation and culture methods. Our lab tries to study the possible relationship between diabetes and microbiota using molecular ecolo
9、gical methods. Some results indicates there is a certain connection between intestinal microflora and the onset and development of diabetes mellitus. Several bacterial strains were revealed directly associated with diabetes. In this study, ICR mice were randomly separated into two groups. One is for
10、 diabetes modeling, and the other is healthy control. After i.v. injection of STZ, diabetic mice were significantly lighter than control ones in weight, showing obvious diabetic symptoms. In addition, several model mice without hyperglycemia(STZ-resistant) was kept in comparison with diabetic ones.
11、Firstly ERIC-PCR fingerprinting technique was used to profile the total DNA of fecal microflora samples and PLS-DA was used to search for the specialized features of different groups . Results indicated the gut flora of 上海交通大学理学硕士学位论文 ABSTRACT diabetic mice(STZ-susceptible) and STZ-resistant mice wa
12、s significantly distinct from that of healthy controls while there is still difference between the STZ-resistant and the STZ-susceptible to some degree. To make out the variance in community structure and distinguished species, 16S rDNA V3 DGGE was adopted. From the resulting pattern, two dominant b
13、ands from controls and the STZ-resistant were observed, however in the same location they were much weakened from diabetic mice. After recovering the targeted DNA from the gel, it is learned by sequencing that two bands represent two lactobacilli respectively. Aiming to investigate the community str
14、ucture of lactobacillus, LAB(Lactic Acid Bacteria) group-specific DGGE was adopted and validated the former observation that two dominant bands from low blood glucose mice were significantly weakened in high blood glucose mice. Sequencing revealed they were Lactobacillus intestinalis and Lactobacill
15、us reuteri. Finally, through principle component analysis of V3 DGGE and LAB DGGE fingerprints, it was determined that these two lactobacilli were the major contributors discriminating the intestinal microflora of two categories of mice while other bacterial communities(bacteriodes, bifidobacteria a
16、nd C.leptum) did not make an obvious difference. Besides, experiment indicated the bifidobacteria in diabetic mice is significantly reduced compared with controls. So it can be concluded that after obvious diabetes shows up, two health-related intestinal bacterial communities(lactobacillis and bifidobacteria) were changed structurally and quantitatively. After that, we contin
