本文關(guān)鍵詞： 代謝組學(xué) 核磁共振 腸道菌群 變性梯度凝膠電泳 林可霉素　出處：《廣州中醫(yī)藥大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：林可霉素干預(yù)大鼠腸道菌群后,基于1NMR的代謝組學(xué)和PCR-DGGE的腸道微生態(tài)分析技術(shù)探討菌群分布、宿主糞便及尿液的代謝變化,進(jìn)而深入研究腸道菌群與宿主代謝的相互關(guān)系。本研究為理解腸道菌群與宿主在代謝水平上的相互作用提供一種系統(tǒng)、整體的方法,為了解菌群在各種代謝疾病發(fā)生、發(fā)展過(guò)程中的作用,提供一種新的研究思路。方法：1.基于PCR-DGGE的林可霉素干預(yù)對(duì)腸道菌群組成的影響首先,我們采用林可霉素誘導(dǎo)腸道菌群失調(diào)模型,利用PCR-DGGE技術(shù)對(duì)大鼠糞樣16SrRNA基因V3區(qū)進(jìn)行擴(kuò)增及變性梯度凝膠電泳,分析林可霉素對(duì)腸道菌群多樣性的影響。同時(shí),采用PCR-DGGE技術(shù)對(duì)擬桿菌和柔嫩梭菌進(jìn)行多樣性分析。2.基于NMR的林可霉素干預(yù)對(duì)糞樣與尿樣代謝的影響采用600 MHz核磁共振儀對(duì)糞便與尿樣進(jìn)行檢測(cè)分析。模式識(shí)別之前,對(duì)所有譜圖進(jìn)行相位、基線、化學(xué)位移校正。然后利用多變量統(tǒng)計(jì)分析方法進(jìn)行分析,包括PCA、PLS-DA和OPLS-DA等。最后將發(fā)現(xiàn)的差異代謝物,用]HDMB、 KEGG進(jìn)行代謝通路分析,解釋差異代謝物的生物學(xué)意義。3.1HNMR數(shù)據(jù)與DGGE數(shù)據(jù)的整合分析通過(guò)NMR和DGGE分析,提取顯著變化的代謝物與菌群條帶,進(jìn)行相關(guān)性分析。結(jié)合OPLS與皮爾森相關(guān)系數(shù)分析結(jié)果,獲得變化的代謝物與變化的DGGE條帶的相關(guān)性。結(jié)果1.經(jīng)過(guò)14天的抗生素處理,林可霉素組菌群結(jié)構(gòu)發(fā)生明顯變化。Barnesiella和Prevotella的數(shù)量急劇下降,而Clostridium cluster ⅩⅣα的含量稍有上升。經(jīng)測(cè)序鑒定的12條V3區(qū)域的16S rRNA基因序列已被GenBank收錄,登錄號(hào)為KP666050-666061。2.林可霉素處理后,馬尿酸、短鏈脂肪酸和初級(jí)膽汁酸等代謝物水平下降,而膽堿、寡糖等的含量明顯增加。這些代謝標(biāo)記物提示林可霉素干預(yù)會(huì)影響腸道菌群對(duì)宿主能量、芳香類氨基酸、膽汁酸的代謝及核酸合成代謝。3.糞便代謝物與腸道中的Escherichia coil、Barnesiella和Prevotella等密切相關(guān)。大腸桿菌屬與膽堿、�；悄懰�、尿嘧啶呈現(xiàn)正相關(guān),而與α-酮異戊酸、甘氨酸、甲酸鹽、丙酸鹽呈負(fù)相關(guān)。存在于正常大鼠腸道中的優(yōu)勢(shì)菌群Barnesiella主要與短鏈脂肪酸,�；悄懰岢尸F(xiàn)正相關(guān),與尿苷酸、異亮氨酸呈負(fù)相關(guān)。結(jié)論：本論文建立了對(duì)腸道菌群-宿主代謝關(guān)系進(jìn)行系統(tǒng)分析的方法,發(fā)現(xiàn)林可霉素干預(yù)不僅影響腸道菌群組成結(jié)構(gòu)及宿主代謝水平,而且腸道菌群中具有重要功能的成員也影響宿主特定的代謝過(guò)程。代謝組學(xué)與腸道微生態(tài)結(jié)合分析的方法,為研究腸道菌群-宿主代謝的相關(guān)性提供重要參考,有效地提供腸道菌群的代謝信息,為研究菌群在各種代謝疾病發(fā)生、發(fā)展過(guò)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，

本文編號(hào)：1528803