本文選題：萊姆病　切入點(diǎn)：伯氏疏螺旋體　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：萊姆病(Lyme disease,LD),亦稱萊姆疏螺旋體病(Lyme borreliosis),是由媒介蜱傳播伯氏疏螺旋體感染引起的自然疫源性人獸共患疾病。代謝組學(xué)(metabonomics)是一門通過(guò)對(duì)某一生物或細(xì)胞中特定生理時(shí)期內(nèi)所有低分子量代謝物的集合進(jìn)行定性、定量分析,并且尋找代謝物與其生理病理變化相對(duì)應(yīng)關(guān)系的學(xué)科。本研究通過(guò)代謝組學(xué)分析尋找LD潛在的生物標(biāo)志物,進(jìn)而了解感染后的致病機(jī)制。1.為確定小鼠腦組織成功感染萊姆病螺旋體并發(fā)生病理變化,將80只BALB/c雌性小鼠隨機(jī)分為4個(gè)實(shí)驗(yàn)組和4個(gè)對(duì)照組,10只/組,實(shí)驗(yàn)組和對(duì)照組分別皮下接種0.2μl萊姆病螺旋體Borrelia garinii SZ株菌液和PBS懸液;在第7d、14d、28d和35d實(shí)驗(yàn)組和對(duì)照組各一組采集腦組織,取少量腦組織分離培養(yǎng),鏡檢發(fā)現(xiàn)有大量螺旋體,說(shuō)明萊姆病螺旋體已成功感染腦組織。同時(shí)制作4個(gè)時(shí)間點(diǎn)的腦組織病理切片,蘇木精-伊紅染色后,結(jié)果顯示實(shí)驗(yàn)組與對(duì)照組相比有:神經(jīng)元體積增大、纖維組織溶解等病理變化,說(shuō)明腦組織感染萊姆病螺旋體后發(fā)生明顯病理變化。然后將樣品做代謝組學(xué)分析。2.為檢驗(yàn)樣本品質(zhì)和摸索樣本氣相色譜-質(zhì)譜連用檢測(cè)技術(shù)最優(yōu)代謝物萃取的方法,先進(jìn)行預(yù)實(shí)驗(yàn)。將小鼠腦組織樣本分8組,每組10例平行樣本,分四個(gè)時(shí)間點(diǎn),通過(guò)代謝物萃取、衍生化處理、樣品檢測(cè)、系統(tǒng)穩(wěn)定性檢測(cè),結(jié)果顯示,樣品品質(zhì)良好,實(shí)驗(yàn)方法、儀器平臺(tái)良好,系統(tǒng)穩(wěn)定性良好。可以開展正式檢測(cè)試驗(yàn)。3.為篩選萊姆病螺旋體Borrelia garinii SZ株感染小鼠腦組織的代謝差異物中潛在生物標(biāo)志物。正式試驗(yàn)將8組樣本經(jīng)過(guò)數(shù)據(jù)預(yù)處理,主成分分析,正交偏最小二乘法-判別分析,差異化合物篩選和鑒定,共篩選出32個(gè)代謝差異物。經(jīng)鑒定這32個(gè)代謝差異物即為本實(shí)驗(yàn)篩選的潛在生物標(biāo)志物。4.為進(jìn)一步了解這32個(gè)潛在生物標(biāo)志物參與的代謝通路。將32個(gè)潛在生物標(biāo)志物通過(guò)本地化數(shù)據(jù)庫(kù)得到了64個(gè)KEGG號(hào),再通過(guò)KEGG pathway網(wǎng)站匹配到相應(yīng)物質(zhì)的代謝通路圖。由于通路圖數(shù)量較多,通過(guò)Cytoscape軟件富集了顯著性排名前10的調(diào)控通路并整合繪制了本實(shí)驗(yàn)所選生物標(biāo)志物參與的通路圖,然后查閱相關(guān)文獻(xiàn)研究各物質(zhì)和通路之間的相互作用。結(jié)果顯示萊姆病螺旋體代謝組學(xué)主要代謝途徑是糖酵解途徑,萊姆病螺旋體僅依靠糖酵解來(lái)產(chǎn)生ATP,該菌不能編碼一些完整的通路來(lái)實(shí)現(xiàn)例如脂肪酸、氨基酸、核酸的生物合成。這為萊姆病螺旋體致病機(jī)制的研究奠定了一定基礎(chǔ)。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S855

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