伽氏疏螺旋体小鼠脑组织感染模型的代谢组学生物标志物筛选

发布时间：2018-03-28 02:20

本文选题：莱姆病　切入点：伯氏疏螺旋体　出处：《甘肃农业大学》2017年硕士论文

【摘要】：莱姆病(Lyme disease,LD),亦称莱姆疏螺旋体病(Lyme borreliosis),是由媒介蜱传播伯氏疏螺旋体感染引起的自然疫源性人兽共患疾病。代谢组学(metabonomics)是一门通过对某一生物或细胞中特定生理时期内所有低分子量代谢物的集合进行定性、定量分析,并且寻找代谢物与其生理病理变化相对应关系的学科。本研究通过代谢组学分析寻找LD潜在的生物标志物,进而了解感染后的致病机制。1.为确定小鼠脑组织成功感染莱姆病螺旋体并发生病理变化,将80只BALB/c雌性小鼠随机分为4个实验组和4个对照组,10只/组,实验组和对照组分别皮下接种0.2μl莱姆病螺旋体Borrelia garinii SZ株菌液和PBS悬液;在第7d、14d、28d和35d实验组和对照组各一组采集脑组织,取少量脑组织分离培养,镜检发现有大量螺旋体,说明莱姆病螺旋体已成功感染脑组织。同时制作4个时间点的脑组织病理切片,苏木精-伊红染色后,结果显示实验组与对照组相比有:神经元体积增大、纤维组织溶解等病理变化,说明脑组织感染莱姆病螺旋体后发生明显病理变化。然后将样品做代谢组学分析。2.为检验样本品质和摸索样本气相色谱-质谱连用检测技术最优代谢物萃取的方法,先进行预实验。将小鼠脑组织样本分8组,每组10例平行样本,分四个时间点,通过代谢物萃取、衍生化处理、样品检测、系统稳定性检测,结果显示,样品品质良好,实验方法、仪器平台良好,系统稳定性良好。可以开展正式检测试验。3.为筛选莱姆病螺旋体Borrelia garinii SZ株感染小鼠脑组织的代谢差异物中潜在生物标志物。正式试验将8组样本经过数据预处理,主成分分析,正交偏最小二乘法-判别分析,差异化合物筛选和鉴定,共筛选出32个代谢差异物。经鉴定这32个代谢差异物即为本实验筛选的潜在生物标志物。4.为进一步了解这32个潜在生物标志物参与的代谢通路。将32个潜在生物标志物通过本地化数据库得到了64个KEGG号,再通过KEGG pathway网站匹配到相应物质的代谢通路图。由于通路图数量较多,通过Cytoscape软件富集了显著性排名前10的调控通路并整合绘制了本实验所选生物标志物参与的通路图,然后查阅相关文献研究各物质和通路之间的相互作用。结果显示莱姆病螺旋体代谢组学主要代谢途径是糖酵解途径,莱姆病螺旋体仅依靠糖酵解来产生ATP,该菌不能编码一些完整的通路来实现例如脂肪酸、氨基酸、核酸的生物合成。这为莱姆病螺旋体致病机制的研究奠定了一定基础。
[Abstract]:Lyme disease, also known as Lyme borreliosisus, is a natural zoonotic disease caused by the transmission of Borrelia burgdorferi by vector ticks. MetabonomicsA metabonomicsA family of natural zoonotic diseases caused by the transmission of Borrelia burgdorferi by a vector tick. MetabonomicsA family of organisms or cells that pass through a specific physiological time in a given organism or cell. A collection of all low molecular weight metabolites during the period, Quantitative analysis and search for the relationship between metabolites and their physiological and pathological changes. This study looked for potential biomarkers of LD by metabonomics analysis. In order to determine the successful infection of Lyme disease in mouse brain and pathological changes, 80 BALB/c female mice were randomly divided into 4 experimental groups and 10 control groups. The experimental group and the control group were inoculated subcutaneously with 0.2 渭 l Borrelia garinii SZ strain and PBS suspension respectively, and the brain tissues were collected from each group on the 7th day, 14 days and 35 days, and a small amount of brain tissue was isolated and cultured, and a large number of spirochetes were found by microscopic examination. At the same time, the pathological sections of brain tissue were made at four time points. After hematoxylin and eosin staining, the results showed that compared with the control group, the neurons in the experimental group were larger than those in the control group. Pathological changes such as fibrinolysis, The results showed that the pathological changes occurred in the brain tissue after infection with Lyme disease, and then the samples were analyzed by metabolomics. 2. To test the quality of the samples and explore the optimal method for the extraction of metabolites by gas chromatography-mass spectrometry. The brain tissue samples of mice were divided into 8 groups, 10 parallel samples in each group, divided into four time points. The samples were extracted by metabolites, treated by derivation, detected by samples, and detected by system stability. The results showed that the quality of the samples was good. The experimental method, the instrument platform is good, In order to screen potential biomarkers in brain tissue of mice infected with Borrelia garinii SZ strain of Lyme disease, eight groups of samples were pretreated with data and analyzed by principal component analysis. Orthogonal partial least squares method-discriminant analysis, screening and identification of differential compounds, A total of 32 metabolic differences were screened out. The 32 metabolic differences were identified as potential biomarkers in this study. 4. In order to further understand the metabolic pathways involved in these 32 potential biomarkers, 32 potential biomarkers were selected. The marker gets 64 KEGG numbers from the localised database, Then the metabolic pathway map of the corresponding substances was matched through the KEGG pathway website. Because of the large number of pathway maps, the top 10 significant regulatory pathways were enriched by Cytoscape software, and the pathway map of the biomarkers selected in this experiment was integrated and drawn. The results showed that the main metabolic pathway of Lyme disease was glycolysis. Lyme disease spirochetes can only be produced by glycolysis, which can not encode some complete pathways for the biosynthesis of fatty acids, amino acids and nucleic acids, which lays a foundation for the study of the pathogenesis of Lyme disease.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S855

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