金黄色葡萄球菌侵袭对人乳腺上皮细胞氨基酸代谢关键酶表达的影响

发布时间：2018-03-11 12:28

本文选题：金黄色葡萄球菌　切入点：氨基酸代谢　出处：《内蒙古大学》2017年硕士论文　论文类型：学位论文

【摘要】：金黄色葡萄球菌(Staphylococcus aureus)是引起哺乳动物感染的关键病原菌之一,易侵入乳腺组织诱发乳腺炎。乳腺炎对人畜造成的负面影响极大,影响乳品的质与量。乳蛋白是乳品重要的组成成分,乳腺上皮细胞中氨基酸的种类、含量直接影响其合成,氨基酸代谢是影响乳蛋白合成的重要因素。细菌侵袭乳腺上皮细胞会影响乳蛋白的合成与分泌,但对于氨基酸代谢的影响与机制目前还不清楚。本研究以人乳腺上皮细胞MCF-10A为模型,利用金黄色葡萄球菌(ATCC27543)侵袭细胞,检测宿主细胞六种氨基酸代谢关键酶及β-酪蛋白的表达情况。六种酶包括天冬氨酸氨基转移酶(AST)、谷胱甘肽硫转移酶(GST)、谷氨酸脱氢酶(GDH)、α-酮戊二酸脱氢酶(OGDH)、鸟氨酸脱羧酶(ODC)和谷氨酸脱羧酶(GAD),分别涉及转氨、脱氨、联合脱氨和脱羧等过程。首先,利用金黄色葡萄球菌侵袭细胞,qPCR检测细胞中氨基酸代谢关键酶基因及β-酪蛋白基因的表达,ELISA检测细胞内外相关基因产物的合成与分泌,western blot检测mTORC1信号通路活性;其次,用肽聚糖刺激人乳腺上皮细胞,同样用qPCR和ELISA方法检测氨基酸代谢关键酶基因及其产物的表达与分泌,western blot检测mTORC1通路及几种转录因子活性;最后,在前两部分实验的基础上探究mTORC1信号通路对相关转录因子活性和氨基酸代谢关键酶表达的调控作用。通过特异性抑制剂Rapamycin及RNA干扰技术降低mTORC1活性,检测金黄色葡萄球菌侵袭与非侵袭条件下氨基酸代谢关键酶及β-酪蛋白基因及其产物的表达变化,并检测几种可能同氨基酸代谢相关的转录因子的活性变化。结果表明:(1)金黄色葡萄球菌能侵入体外培养的人乳腺上皮细胞内,随着侵袭过程的进行,细胞内氨基酸代谢关键酶含量呈先上升而后下降的趋势,细菌侵袭8h后,β-酪蛋白的合成量与分泌量显著下降(p0.001)。同时,细菌侵袭过程中mTOR信号通路的活性也呈现先上升而后下降的趋势;(2)肽聚糖刺激MCF-10A细胞,诱导细胞内氨基酸代谢关键酶表达,含量显著上升(p0.001),而对β-酪蛋白的合成与分泌没有显著影响(p0.05)。同时mTOR信号通路和几种转录因子的活性增强;(3)Rapamycin能够显著抑制上述细菌侵袭和肽聚糖刺激对氨基酸代谢关键酶基因的诱导表达(p0.01),导致氨基酸代谢关键酶在细胞内的含量显著下降(p0.01),β-酪蛋白的合成与分泌也受到抑制(p0.01),mTORC1信号通路和相关转录因子的活性均有不同程度的下降;(4)通过靶向siRNA沉默mTORC1关键组分Raptor编码基因,进而抑制mTORC1信号通路,所得结果与上述Rapamycin处理结果一致,印证了 mTORC1信号通路对氨基酸代谢关键酶和β-酪蛋白的合成与分泌具有调控作用。综上所述,金黄色葡萄球菌可以侵袭进入人乳腺上皮细胞,影响宿主细胞内六种氨基酸代谢关键酶与β-酪蛋白的合成与分泌。随着细菌侵袭时间的延长,mTOR信号通路的活性有先上升后下降的趋势,细胞内氨基酸代谢关键酶的表达也呈先上升后稍有下降的趋势,β-酪蛋白的合成与分泌减弱。金黄色葡萄球菌肽聚糖(PGN)可以诱导六种氨基酸代谢关键酶的表达,激活mTORC1信号通路及相关转录因子。mTORC1信号通路介导细菌侵袭和PGN刺激信号,可能通过对转录因子NF-κB,STAT1,STAT3,SREBP1和PPARγ活性的调控实现对GST、AST、GDH、OGDH、ODC和GAD及β-酪蛋白的合成与分泌的调控。实验所得结果为进一步探究mTORC1信号通路对氨基酸代谢的调控作用及其机制提供了基础数据。
[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.

【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S857.26

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