林可霉素干预大鼠“肠道菌群—宿主代谢”的相关性研究

发布时间：2018-02-24 04:06

  本文关键词： 代谢组学 核磁共振 肠道菌群 变性梯度凝胶电泳 林可霉素　出处：《广州中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：林可霉素干预大鼠肠道菌群后,基于1NMR的代谢组学和PCR-DGGE的肠道微生态分析技术探讨菌群分布、宿主粪便及尿液的代谢变化,进而深入研究肠道菌群与宿主代谢的相互关系。本研究为理解肠道菌群与宿主在代谢水平上的相互作用提供一种系统、整体的方法,为了解菌群在各种代谢疾病发生、发展过程中的作用,提供一种新的研究思路。方法：1.基于PCR-DGGE的林可霉素干预对肠道菌群组成的影响首先,我们采用林可霉素诱导肠道菌群失调模型,利用PCR-DGGE技术对大鼠粪样16SrRNA基因V3区进行扩增及变性梯度凝胶电泳,分析林可霉素对肠道菌群多样性的影响。同时,采用PCR-DGGE技术对拟杆菌和柔嫩梭菌进行多样性分析。2.基于NMR的林可霉素干预对粪样与尿样代谢的影响采用600 MHz核磁共振仪对粪便与尿样进行检测分析。模式识别之前,对所有谱图进行相位、基线、化学位移校正。然后利用多变量统计分析方法进行分析,包括PCA、PLS-DA和OPLS-DA等。最后将发现的差异代谢物,用]HDMB、 KEGG进行代谢通路分析,解释差异代谢物的生物学意义。3.1HNMR数据与DGGE数据的整合分析通过NMR和DGGE分析,提取显著变化的代谢物与菌群条带,进行相关性分析。结合OPLS与皮尔森相关系数分析结果,获得变化的代谢物与变化的DGGE条带的相关性。结果1.经过14天的抗生素处理,林可霉素组菌群结构发生明显变化。Barnesiella和Prevotella的数量急剧下降,而Clostridium cluster ⅩⅣα的含量稍有上升。经测序鉴定的12条V3区域的16S rRNA基因序列已被GenBank收录,登录号为KP666050-666061。2.林可霉素处理后,马尿酸、短链脂肪酸和初级胆汁酸等代谢物水平下降,而胆碱、寡糖等的含量明显增加。这些代谢标记物提示林可霉素干预会影响肠道菌群对宿主能量、芳香类氨基酸、胆汁酸的代谢及核酸合成代谢。3.粪便代谢物与肠道中的Escherichia coil、Barnesiella和Prevotella等密切相关。大肠杆菌属与胆碱、牛磺胆酸、尿嘧啶呈现正相关,而与α-酮异戊酸、甘氨酸、甲酸盐、丙酸盐呈负相关。存在于正常大鼠肠道中的优势菌群Barnesiella主要与短链脂肪酸,牛磺胆酸呈现正相关,与尿苷酸、异亮氨酸呈负相关。结论：本论文建立了对肠道菌群-宿主代谢关系进行系统分析的方法,发现林可霉素干预不仅影响肠道菌群组成结构及宿主代谢水平,而且肠道菌群中具有重要功能的成员也影响宿主特定的代谢过程。代谢组学与肠道微生态结合分析的方法,为研究肠道菌群-宿主代谢的相关性提供重要参考,有效地提供肠道菌群的代谢信息,为研究菌群在各种代谢疾病发生、发展过程中的作用提供新的研究思路。
[Abstract]:Objective: to investigate the distribution of microflora, the metabolic changes in feces and urine of rats with lincomycin intervention, based on 1NMR metabolomics and PCR-DGGE microecological analysis. This study provides a systematic and holistic approach to understand the interactions between intestinal flora and host at metabolic level, and to understand the occurrence of microflora in various metabolic diseases. The role of lincomycin in the development process provides a new way of thinking. Methods: 1.The effect of lincomycin intervention based on PCR-DGGE on the composition of intestinal flora. Firstly, we used lincomycin to induce intestinal flora imbalance model. The V3 region of 16s rRNA gene in rat feces was amplified by PCR-DGGE and denaturing gradient gel electrophoresis was used to analyze the effect of lincomycin on the diversity of intestinal flora. Diversity analysis of Bacteroides tenella and Clostridium tenella by PCR-DGGE. 2. Effects of lincomycin intervention based on NMR on fecal and urine metabolism. Fecal and urine samples were detected and analyzed by 600 MHz NMR. Before pattern recognition, The phase, baseline and chemical shift of all spectra were corrected. Then the multivariate statistical analysis was used, including PCAPS-DA and OPLS-DA, etc. Finally, the differential metabolites were analyzed by] HDMBand KEGG. To explain the biological significance of differential metabolites .3.1H NMR data and the integration analysis of DGGE data. By NMR and DGGE analysis, the significantly changed metabolites and microbial bands were extracted, and the correlation analysis was carried out. The results of correlation coefficient analysis between OPLS and Pearson were combined. Results 1. After 14 days of antibiotic treatment, the structure of lincomycin group flora changed significantly. The number of Prevotella and Prevotella decreased sharply. 2. However, the content of Clostridium cluster 鈪，

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