通讨肠道微生物群落结构演替—初探益生菌改善便秘的机制

发布时间：2018-05-06 10:32

本文选题：慢传输型便秘 + 肠道微生物群落结构　；参考：《昆明理工大学》2017年硕士论文

【摘要】：慢传输型便秘(slow transit constipation,STC)是临床常见的慢性顽固型便秘,占便秘发病率的25%。其临床表现顽固,综合保守治疗效果不佳。由于临床滥用泻剂,导致便秘的进行性加重,最终导致药物失效只能实施手术治疗。由于该疾病的发病机制至今尚不明确,给临床防治带来极大困难。本研究使用分子生物学方法结合高通量测序分析慢传输型便秘及益生菌制剂缓解慢传输型便秘过程中粪便样品中的微生物群落结构,并对构建的慢传输型便秘小鼠生理指标进行分析,研究益生菌混合发酵酸奶对慢传输型便秘的治疗效果,以期为临床诊断及治疗提供理论依据。小鼠及便秘患者实验结果表明,自主配方三菌酸奶可明显缓解慢传输型便秘症状。通过对小鼠生理指标的检测,可以看出三菌酸奶组小鼠的小肠推进率、首次排黑便时间均优于其他组,且三菌酸奶可明显逆转小鼠的便秘症状。而通过小鼠粪便样品中短链脂肪酸(short-chain fatty acids,SCFAs)的检测可以看出三菌酸奶有明显的降低能量供应的作用。从OTU数目、ACE和Chaol指数来看,随着喂食酸奶天数的增加,小鼠粪便样品及慢传输型便秘患者粪便样品中的细菌多样性显著降低,而小鼠在喂食便秘药物洛哌丁胺后,两菌酸奶组小鼠OTU数目与健康对照组增加且无显著差别,而三菌酸奶组与便秘模型组降低。慢传输型便秘患者粪便样品中的细菌多样性显著降低,这说明便秘导致肠道中的有害微生物增加,而可能导致其他一些微生物的生长受到抑制甚至死亡,喂食益生菌混合发酵酸奶后,有益微生物在肠道内大量定植,抑制了有害微生物的生长,从而使粪便样品中细菌的多样性呈现下降的趋势。在小鼠实验中,比较不同组所有小鼠的细菌群落结构显示,在门的分类水平上,四个组的小鼠粪便样品中主要的细菌为Bacteroidetes、Firmicutes和Proteobacteria。饮用两菌酸奶组小鼠粪便样品中Firmicutes与Proteobacteria的量明显低于其他组,而Bacteroidetes所占序列比例更高。随着实验天数的增加,各组小鼠粪便样品中的微生物群落发生着明显的改变。对不同组别的小鼠按处理时间进行微生物群落分析可以看出,小鼠粪便样品中Candidatusfamily ratAN060301C、Rikenellacae随着喂食小鼠酸奶天数的增加而增加,而Lachnospiraceae、Desulfovibrionaceae 与 Carnobacteriaceae 是减少的,其之所以在粪便样品中增加或减少是由于其在肠道内的定植发生改变。UPGMA(unweighted pair-group method with arithmetic means)聚类和主成分分析(principal components analysis,PCA)都表明,不管何种方法,所得到的细菌群落聚类结果大致相同,喂食益生菌制剂前小鼠粪便样品中的细菌群落结构分布不均匀,无显著相关性,而喂食益生菌制剂后小鼠粪便样品中细菌群落结构较为相似,且三菌酸奶组比两菌酸奶组更为相似。比较所有便秘患者饮用益生菌混合发酵酸奶前后粪便样品中细菌群落结构,可以看出,在门的分类水平上,饮用益生菌混合发酵酸奶前粪便样品中的细菌多样性高于饮用后,而其中有近一半的序列属于Bacteroidetes和Firmicutes,Proteobacteria和Actinobacteria序列所占比例较少。在属的分类水平上,饮用益生菌混合发酵酸奶前粪便样品中的细菌多样性高于饮用后,但两个样品中优势属所占的序列百分比并无明显差异,均为Bacreroides和Faecalibacterium。通过对每个患者饮用益生菌混合发酵酸奶前后粪便样品中细菌的主要属进行的分析和比对可以看出,绝大多数患者饮用益生菌混合发酵酸奶前后粪便样品中的优势菌均为 Bacteroides,大多数是 Faecalibacterium。研究慢传输型便秘患者饮用益生菌混合发酵酸奶前后粪便样品微生物群落结构组成和演替规律,结合小鼠便秘模型试验,寻找引起便秘及益生菌混合发酵酸奶缓解便秘过程中的关键微生物,为便秘的临床治疗提供有力的新的治疗手段。
[Abstract]:Slow transit constipation (STC) is a common chronic and stubborn constipation in clinic. The clinical manifestation of 25%. in the incidence of constipation is obstinate, and the effect of comprehensive conservative treatment is not good. Because of the clinical abuse of laxatives, the progressive constipation is aggravated and the drug failure can only be performed by operation. The system is still not clear to date and brings great difficulties to clinical prevention and treatment. This study uses molecular biological methods and high throughput sequencing to analyze slow transmission constipation and probiotics to alleviate the microbial community structure in the process of slow transmission constipation, and analyze the physiological indexes of the slow transmission type constipation mice. The therapeutic effect of probiotic mixed fermented yogurt on slow transit constipation was provided in order to provide theoretical basis for clinical diagnosis and treatment. The experimental results of mice and constipation patients showed that the spontaneous formulation of three bacteria yogurt could obviously relieve the symptoms of slow transmission constipation. By testing the physiological indexes of mice, the small intestine of the three bacteria yogurt mice could be seen. The rate of intake was better than the other groups for the first time, and the three bacteria yogurt could obviously reverse the symptoms of constipation in mice. Through the detection of short-chain fatty acids, SCFAs in the fecal samples of mice, it was found that three bacteria yogurt had a significant reduction in energy supply. From the number of OTU, ACE and Chaol index, the feed was fed with feeding. The increase in the number of yogurt days, the bacterial diversity in the fecal samples of the mice and the slow transit constipation patients decreased significantly, while in the mice fed with the constipation drug luperidine, the number of OTU in the two bacteria yogurt mice increased and no significant difference was found, while the three bacteria yogurt group and the constipation model group decreased. The bacterial diversity in the fecal samples is significantly reduced, which indicates that constipation leads to the increase of harmful microbes in the intestinal tract, which may lead to the inhibition or even death of some other microorganisms. After feeding the probiotics to the mixed fermented yogurt, the beneficial microbes colonize the intestinal tract and inhibit the growth of harmful microorganisms, thus causing feces. In the mice experiment, the bacterial community structure of all the mice in the different groups showed that the main bacteria in the four groups of mice fecal samples were Bacteroidetes, Firmicutes and Proteobacteria. in the fecal samples of the two bacteria yogurt mice, and Firmicutes and Pro in the fecal samples of the mice. The amount of teobacteria was significantly lower than that of the other groups, and the proportion of Bacteroidetes was higher. With the increase of the experiment days, the microbial community in the fecal samples of each group changed obviously. The microorganism community analysis of the mice in different groups could be seen, and the Candidatusfamily ratA in the mice feces samples. N060301C, Rikenellacae increased with the increase in the number of yoghurt days in feeding mice, while Lachnospiraceae, Desulfovibrionaceae and Carnobacteriaceae were reduced, and the reason for the increase or decrease in fecal samples was due to the change of.UPGMA (unweighted pair-group method with arithmetic means) clustering in the intestinal tract. Principal components analysis (PCA) and the principal component analysis (analysis) showed that the results of the bacterial community clustering were almost the same regardless of the method. The bacterial community structure in the fecal samples of the pre feeding probiotic preparation was not uniform, and the bacterial community structure in the fecal samples of the mice after feeding probiotics was fed. The three bacteria yogurt group was more similar than the two bacteria yogurt group. Compared with the bacteria community structure in the fecal samples before and after the mixed fermented yogurt of all constipation patients, it can be seen that the diversity of the bacteria in the fecal samples before the mixed fermentation of the yogurt was higher than that in the faecal samples before the mixed fermentation of the yogurt. Half of the sequences belong to Bacteroidetes and Firmicutes, and the proportion of Proteobacteria and Actinobacteria sequences is less. In the classification level of the genus, the bacterial diversity in the fecal samples of the mixed fermented yoghurt before drinking is higher than that after drinking, but there is no significant difference in the percentage of the dominant genera in the two samples, all of which are Bacreroid By analyzing and comparing the main genera of the bacteria in the fecal samples before and after the mixed fermented yogurt of each patient, ES and Faecalibacterium. can see that the dominant bacteria in the fecal samples of the overwhelming majority of patients who drank the probiotic mixed fermented yogurt were Bacteroides, most of which were the slow transmission of Faecalibacterium.. The microbial community structure and succession law of the fecal sample before and after the mixed fermentation of probiotics in the constipation of the constipation patients, combined with the mouse constipation model test, to find the key microbes in the process of constipation causing constipation and probiotic mixed fermented yogurt to relieve constipation, and provide a powerful new therapeutic means for the clinical treatment of constipation.

【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62

【参考文献】