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  本文選題：金黃色葡萄球菌　切入點：氨基酸代謝　出處：《內蒙古大學》2017年碩士論文　論文類型：學位論文

【摘要】：金黃色葡萄球菌(Staphylococcus aureus)是引起哺乳動物感染的關鍵病原菌之一,易侵入乳腺組織誘發(fā)乳腺炎。乳腺炎對人畜造成的負面影響極大,影響乳品的質與量。乳蛋白是乳品重要的組成成分,乳腺上皮細胞中氨基酸的種類、含量直接影響其合成,氨基酸代謝是影響乳蛋白合成的重要因素。細菌侵襲乳腺上皮細胞會影響乳蛋白的合成與分泌,但對于氨基酸代謝的影響與機制目前還不清楚。本研究以人乳腺上皮細胞MCF-10A為模型,利用金黃色葡萄球菌(ATCC27543)侵襲細胞,檢測宿主細胞六種氨基酸代謝關鍵酶及β-酪蛋白的表達情況。六種酶包括天冬氨酸氨基轉移酶(AST)、谷胱甘肽硫轉移酶(GST)、谷氨酸脫氫酶(GDH)、α-酮戊二酸脫氫酶(OGDH)、鳥氨酸脫羧酶(ODC)和谷氨酸脫羧酶(GAD),分別涉及轉氨、脫氨、聯(lián)合脫氨和脫羧等過程。首先,利用金黃色葡萄球菌侵襲細胞,qPCR檢測細胞中氨基酸代謝關鍵酶基因及β-酪蛋白基因的表達,ELISA檢測細胞內外相關基因產物的合成與分泌,western blot檢測mTORC1信號通路活性;其次,用肽聚糖刺激人乳腺上皮細胞,同樣用qPCR和ELISA方法檢測氨基酸代謝關鍵酶基因及其產物的表達與分泌,western blot檢測mTORC1通路及幾種轉錄因子活性;最后,在前兩部分實驗的基礎上探究mTORC1信號通路對相關轉錄因子活性和氨基酸代謝關鍵酶表達的調控作用。通過特異性抑制劑Rapamycin及RNA干擾技術降低mTORC1活性,檢測金黃色葡萄球菌侵襲與非侵襲條件下氨基酸代謝關鍵酶及β-酪蛋白基因及其產物的表達變化,并檢測幾種可能同氨基酸代謝相關的轉錄因子的活性變化。結果表明:(1)金黃色葡萄球菌能侵入體外培養(yǎng)的人乳腺上皮細胞內,隨著侵襲過程的進行,細胞內氨基酸代謝關鍵酶含量呈先上升而后下降的趨勢,細菌侵襲8h后,β-酪蛋白的合成量與分泌量顯著下降(p0.001)。同時,細菌侵襲過程中mTOR信號通路的活性也呈現(xiàn)先上升而后下降的趨勢;(2)肽聚糖刺激MCF-10A細胞,誘導細胞內氨基酸代謝關鍵酶表達,含量顯著上升(p0.001),而對β-酪蛋白的合成與分泌沒有顯著影響(p0.05)。同時mTOR信號通路和幾種轉錄因子的活性增強;(3)Rapamycin能夠顯著抑制上述細菌侵襲和肽聚糖刺激對氨基酸代謝關鍵酶基因的誘導表達(p0.01),導致氨基酸代謝關鍵酶在細胞內的含量顯著下降(p0.01),β-酪蛋白的合成與分泌也受到抑制(p0.01),mTORC1信號通路和相關轉錄因子的活性均有不同程度的下降;(4)通過靶向siRNA沉默mTORC1關鍵組分Raptor編碼基因,進而抑制mTORC1信號通路,所得結果與上述Rapamycin處理結果一致,印證了 mTORC1信號通路對氨基酸代謝關鍵酶和β-酪蛋白的合成與分泌具有調控作用。綜上所述,金黃色葡萄球菌可以侵襲進入人乳腺上皮細胞,影響宿主細胞內六種氨基酸代謝關鍵酶與β-酪蛋白的合成與分泌。隨著細菌侵襲時間的延長,mTOR信號通路的活性有先上升后下降的趨勢,細胞內氨基酸代謝關鍵酶的表達也呈先上升后稍有下降的趨勢,β-酪蛋白的合成與分泌減弱。金黃色葡萄球菌肽聚糖(PGN)可以誘導六種氨基酸代謝關鍵酶的表達,激活mTORC1信號通路及相關轉錄因子。mTORC1信號通路介導細菌侵襲和PGN刺激信號,可能通過對轉錄因子NF-κB,STAT1,STAT3,SREBP1和PPARγ活性的調控實現(xiàn)對GST、AST、GDH、OGDH、ODC和GAD及β-酪蛋白的合成與分泌的調控。實驗所得結果為進一步探究mTORC1信號通路對氨基酸代謝的調控作用及其機制提供了基礎數(shù)據(jù)。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is one of the key pathogenic bacteria in mammalian infection, easily invade breast tissue induced by mastitis. The negative effects on human and animal mastitis caused great influence, the quantity and quality of dairy products. Dairy milk protein is a important component, kinds of amino acids in mammary epithelial cells, directly affect the synthesis of content amino acid metabolism, is an important factor affecting milk protein synthesis. The bacterial invasion of breast epithelial cells will affect the synthesis and secretion of milk protein, but the effect and mechanism of amino acid metabolism is still unclear. In this study, human mammary epithelial cells in MCF-10A model with Staphylococcus aureus (ATCC27543) cell invasion, expression detection of host cells of six key enzymes of amino acid metabolism and beta casein. Six enzymes including aspartate aminotransferase (AST), glutathione S-transferase (G Gu Guanggan ST), glutamate dehydrogenase (GDH), alpha ketoglutarate dehydrogenase (OGDH), ornithine decarboxylase (ODC) and glutamate decarboxylase (GAD), were involved in transamination, deamination and decarboxylation, deamination of combined process. First, using Staphylococcus aureus invasion cells, qPCR was used to detect the expression of key enzymes amino acid metabolism genes and beta casein gene, synthesis and secretion of ELISA cells and related gene products detection, Western detection of blot mTORC1 signaling pathway activity; secondly, with peptidoglycan stimulation in human mammary epithelial cells, the same with the expression of qPCR and ELISA for the detection of key enzyme genes and their products of amino acid metabolism and secretion of western, blot detection of mTORC1 pathway and several transcription factor activity; finally, explore the role of mTORC1 signaling pathway on the expression of key enzyme related transcription factor activity and amino acid metabolism in the first two parts based on the experiment of the specific. Inhibitor of Rapamycin and RNA interference technology to reduce the activity of mTORC1, expression of Staphylococcus aureus invasion and non invasion under the condition of amino acid metabolism enzymes and beta casein gene and its products, and to detect several possible changes of activity of transcription factor related with the metabolism of amino acids. The results show that: (1) Staphylococcus aureus can invasion of cultured human mammary epithelial cells, with the process of invasion and intracellular content of key enzymes of amino acid metabolism was first increased and then decreased trend of bacterial invasion of 8h, the amount of beta casein synthesis and secretion was significantly decreased (p0.001). At the same time, bacterial invasion of mTOR signaling pathway in the process of activity also presented increased first and then decreased; (2) the peptidoglycan stimulation of MCF-10A cells, induce the expression of key enzymes of amino acid metabolism in cells, significantly increased the content of (p0.001), and the synthesis of beta casein No significant effect and secretion (P0.05). At the same time, the mTOR signal transduction pathway and several transcription factor activity increased; (3) Rapamycin can significantly inhibit the bacterial invasion and peptidoglycan induced expression of key enzymes of amino acid metabolism gene (P0.01), lead content of key enzymes of amino acid metabolism in cells decreased significantly (P0.01). Synthesis and secretion of beta casein inhibited (P0.01), the activity of both mTORC1 signaling and transcription factors related to different degrees of decline; (4) Raptor gene encoding siRNA to silence mTORC1 key group through the target, thereby inhibiting the mTORC1 signaling pathway, and the results of the Rapamycin treatment results, has confirmed the regulation effect of synthesis and secretion of mTORC1 signal pathway on amino acid metabolism enzymes and beta casein. In conclusion, Staphylococcus aureus can invade into human mammary epithelial cells, persistent effect Synthesis and secretion of the main cells of six key enzymes of amino acid metabolism and beta casein. With prolonging the time of bacterial invasion, the activity of the mTOR signaling pathway was first increased and then decreased, the expression of intracellular amino acid metabolism key enzyme also increased and then slightly decreased, the synthesis and secretion of beta casein weakened. Staphylococcus aureus peptidoglycan (PGN) can induce the expression of six kinds of amino acid metabolism key enzyme, mTORC1 signaling pathways and transcription factors related to.MTORC1 signaling pathway mediated invasion and PGN bacteria stimulation signal activation, probably via the transcription factor NF- kappa B, STAT1, STAT3, SREBP1 and PPAR activity of regulation on the implementation of the gamma GST, AST, GDH, OGDH, and the regulation of the synthesis of ODC and secretion of GAD and beta casein. The experimental results provide basic data for further exploring the regulatory role and mechanism of mTORC1 signaling pathway on the metabolism of amino acids.

【學位授予單位】：內蒙古大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S857.26

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相關期刊論文 前2條

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本文編號：1598169