慢传输型便秘动物模型的建立及肠道微生物多样性分析

发布时间：2018-02-21 09:35

本文关键词： 肠道微生物慢传输型便秘动物模型粪菌移植 16S rDNA 高通量测序　出处：《南京大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着分子生物学、微生物学以及生物信息学技术的发展,有关肠道微生物与人类疾病相关性的研究越来越深入。慢传输型便秘作为一类发病率高、诊疗复杂且容易反复的胃肠道疾病,其发病机制尚未完全清楚,近年来肠道微生物领域取得的突破为慢传输型便秘提供了新的诊疗策略。基于慢传输型便秘患者体内肠道微生物群落组成结构的变化,本研究试图采用粪菌移植的方式构建慢传输型便秘的动物模型,并对其肠道微生物的多样性进行分析,从而探讨出肠道微生物在慢传输型便秘发生发展过程中所发挥的作用。粪菌活力是造模成功与否的关键,因此研究首先采用LIVE/DEAD细菌死活染色技术,对造模所需的粪便样品进行活力分析。根据临床上采集粪便的实际情况,选取两种粪菌保存方法,即新鲜粪便直接-80℃保存以及新鲜粪便制成30%甘油粪菌液后-8℃保存,分别对两种保存方法下不同时间段内的粪菌活力进行定性分析和定量分析,结果显示粪便直接保存在-8℃中能够在短时间内保持粪菌的活力,但连续两个月之后活菌比例下降至68.3%,明显低于含30%甘油粪菌液的活菌比例(76%)。由于粪菌移植的时间长达2个月,且临床上收集粪便具有较高的不确定性,因此实验中将收集的新鲜粪便预先冻存在-80°C中,12h内制成含30%甘油粪菌液再进行长期保存。按照供体选择标准选取1例健康人和2例便秘患者,分别将其粪便制成粪菌液后,通过灌胃的方式移植给C57BL/6鼠,为降低小鼠体内原有的肠道微生物负荷,提高粪菌移植的效率,C57BL/6鼠在粪菌移植之前进行4周的抗生素处理。此外,本研究还采用了复方地芬诺酯法建立慢传输型便秘模型,以此作为阳性对照。结果显示,抗生素处理后的小鼠盲肠增大,可培养的肠道微生物数量显著降低,表现出无菌鼠的特征。与复方地芬诺酯法造模的小鼠类似,移植便秘患者粪菌小鼠的24h排便量、粪便含水量、Cajal间质细胞的数量于明显低于对照组,且粪菌移植法造模的小鼠结肠内出现气体滞留的现象,与慢传输型便秘的临床症状相似,表明造模成功。提取各组小鼠的粪便DNA,利用16S rDNA二代测序技术分析肠道微生物的群落组成结构,结果显示抗生素处理后的小鼠肠道微生物数量及物种丰富程度显著降低,8周的粪菌移植能够重建小鼠体内的肠道微生物,物种的组成及丰度与供体相比呈现出较高的相似度,达到"人源化"水平。此外,便秘小鼠的肠道微生物与空白对照组以及移植健康人粪菌的小鼠相比有着明显的差别,利用LEfSe寻找差异显著的物种,分析表明小鼠肠道中的优势菌群由拟杆菌属(Barteroides)转变为毛螺菌属(Lachnospira)、δ 变形菌(Deltaproteobacteria)、脱硫弧菌属(Desulfovibrio)、Akkermansia属以及疣微菌属(Verrucomicrobia)。基于此研究结果,未来可以人为地构建出一套肠道菌群进行慢传输型便秘模型的建立,从而规范建模的方法以及模型的判定标准,以便更好地研究肠道微生物与宿主相互作用的具体机制,为慢传输型便秘发病机制研究以及治疗手段构建奠定基础。
[Abstract]:With the development of molecular biology, microbiology and bioinformatics technology, research about the correlation between intestinal microflora and human disease more and more deeply. The slow transit constipation as a kind of high incidence, diagnosis and treatment of complex and prone to recurrent gastrointestinal diseases, its pathogenesis has not yet entirely clear, achieved in the field of gut microbes in recent years breakthrough to slow constipation provides a new therapeutic strategy. Changes in structure of slow transit constipation in patients with intestinal microbial community based on, this study attempts to construct the animal model of slow transit constipation by fecal bacteria transplantation method, and the diversity of gut microbes were analyzed, so as to explore its occurrence and development process of intestinal microflora in the slow transit constipation. Fecal bacteria activity is the key to the success of the model, so the research firstly by LIVE/DEAD bacteria. Dyeing technology, analysis of the model of energy required for fecal samples. According to the actual situation of clinical stool samples, select two kinds of fecal bacteria preservation methods, namely direct stool -80 C storage and fresh stool made of 30% fecal bacteria liquid after glycerol stored at -8, fecal bacteria activity of the two kinds of different preservation methods the time period of the qualitative analysis and quantitative analysis, the results revealed that saved directly to keep fecal bacteria in a short period of time at -8 DEG C and vitality, but after two months the proportion of living bacteria decreased to 68.3%, significantly lower than the proportion of living bacteria of fecal bacteria containing 30% glycerol (76%). The fecal bacteria transplantation the time for 2 months, and the clinical collected stool with high uncertainty, so the experiment will collect the fresh feces of pre frozen -80 degrees C, 12h made of fecal bacteria containing 30% glycerol solution for long-term preservation. According to donor selection criteria A total of 1 healthy people and 2 cases of patients with constipation, the feces were made of fecal bacteria liquid after transplantation through gavage to C57BL/6 rats, mice in order to reduce the original intestinal microbial load, improve the efficiency of fecal bacteria transplantation, C57BL/6 rats were born 4 weeks of anti hormone treatment before FMT. In addition, this study also uses compound diphenoxylate method to establish the model of slow transit constipation, as a positive control. The results showed that the increase of antibiotic treated mice cecum, the numbers of culturable gut microbes decreased significantly, characterized by sterile rats. With compound diphenoxylate of modeling mice is similar to that of 24h stools fecal transplantation patients with constipation bacteria in mice, fecal water content, Cajal and Leydig cell numbers to significantly lower than the control group, gas retention phenomenon of mouse colon and fecal bacteria transplantation model in clinical and slow transit constipation The symptoms are similar, indicating that the animal model successfully. DNA extracted from feces of mice in each group, analysis of the composition and structure of intestinal microbial community using 16S rDNA two generation sequencing technology, the results showed the antibiotic treated mice intestinal microbial quantity and species richness decreased, fecal bacteria 8 weeks of transplantation to the intestinal microbial reconstruction in mice, composition and abundance compared with donor species showed high similarity to "humanized" level. In addition, intestinal microflora and the blank control group and constipation mice transplanted healthy human fecal bacteria compared with the obvious difference, using LEfSe to find significant differences between species, analysis showed that the dominant bacteria in the intestinal tract of mice by fitting group bacillus (Barteroides) into Mao Spirillum (Lachnospira), delta Proteobacteria (Deltaproteobacteria), Desulfovibrio (Desulfovibrio), Akkermansia and Verrucomicrobium (Ver Rucomicrobia). Based on the results of the research, the future can be artificially constructed to establish a set of intestinal microflora of slow transit constipation model, thus the standard modeling method and standard model, in order to better study the specific mechanism of the gut microbiota and host interactions, lay a foundation for the construction research of the pathogenesis of slow transit constipation as well as treatment.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62;R-332

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