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  本文關鍵詞： CTSD 相互作用蛋白 免疫共沉淀 質譜分析 生物信息學分析　出處：《南華大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的： 篩選并鑒定鼻咽癌細胞中組織蛋白酶D (Cathepsin D, CTSD)的相互作用蛋白，初步了解CTSD在鼻咽癌侵襲轉移中的作用及機制。方法： 收集高轉移鼻咽癌5-8F細胞的總蛋白，采用免疫共沉淀結合凝膠電泳分離CTSD相互作用蛋白，應用電噴霧四級桿飛行時間串聯(lián)質譜鑒定相互作用蛋白，結合基因本體論、功能聚類、信號通路、蛋白質相互作用網(wǎng)絡分析等生物信息學方法綜合分析CTSD相互作用蛋白，采用免疫共沉淀結合Western blot方法對CTSD相互作用蛋白EGFR、HSP90A進行驗證。結果1、采用免疫共沉淀結合質譜方法篩選并鑒定了143個CTSD相互作用蛋白。2、將143個CTSD相互作用蛋白進行功能聚類分析顯示：其中70個CTSD相互作用蛋白根據(jù)功能聚為12類，主要涉及到以下幾個方面：跨膜運輸、細胞骨架、氧化磷酸化、蛋白質合成、細胞凋亡、信號轉到、氧化還原酶活性、分子伴侶、糖代謝等。另外73個CTSD相互作用蛋白未參與聚類。3、將143個CTSD相互作用蛋白進行GO分析，結果如下：GO_BP分析顯示CTSD相互作用蛋白生物學過程主要涉及到應激反應、負調控、代謝過程、運輸過程、蛋白定位及轉運過程；GO_MF分析顯示CTSD相互作用蛋白分子功能主要涉及到蛋白結合、催化活性、嘌呤核苷酸綁定、核苷酸結合、細胞骨架、氧化還原酶活性、結構分子活性等；GO_CC分析顯示CTSD相互作用蛋白亞細胞定位于膜、細胞器、囊泡、高分子復合物中等。4、KEGG和Biocarta信號通路分析顯示：143個CTSD相互作用蛋白涉及2條Biocarta信號通路包括低氧誘導因子相關信號通路，端粒、端粒酶、細胞老化；9條KEGG信號通路：氧化磷酸化、丙酮酸代謝、糖酵解信號通路、粘附連接、抗原處理和遞呈、丙酸代謝、核糖體信號通路、磷酸戊糖信號通路。5、蛋白質相互作用網(wǎng)絡分析顯示：EGFR、HSP90A蛋白與CTSD形成相互作用組。6、應用免疫共沉淀結合Western blot顯示EGFR、HSP90A與CTSD結合，驗證了質譜結果的可靠性。 結論1、本研究采用免疫共沉淀結合質譜分析在鼻咽癌細胞中鑒定了143個CTSD的相互作用蛋白，為進一步研究CTSD蛋白的功能提供了實驗依據(jù)。2、生物信息學綜合分析發(fā)現(xiàn)，，EGFR、HSP90A蛋白與CTSD在鼻咽癌細胞中形成相互作用組，可能在鼻咽癌侵襲轉移中發(fā)揮重要作用。3、應用免疫共沉淀結合Western blot對其中2個CTSD相互作用蛋白質EGFR、HSP90A進行了驗證，與質譜結果一致，提示質譜結果的可靠性。
[Abstract]:Objective:. To screen and identify the interacting proteins of cathepsin D Cathepsin D (CTSDs) in nasopharyngeal carcinoma cells, and to understand the role and mechanism of CTSD in the invasion and metastasis of nasopharyngeal carcinoma. The total proteins of 5-8F cells with high metastasis were collected. The CTSD interacting proteins were separated by immunoprecipitation and gel electrophoresis. The interacting proteins were identified by electrospray four-step time-of-flight mass spectrometry. Bioinformatics methods, such as functional clustering, signal pathway and protein interaction network analysis, are used to analyze CTSD interacting proteins. The CTSD interaction protein EGFRN HSP90A was verified by immunoprecipitation and Western blot. Results 1. 143 CTSD interacting proteins were screened and identified by immunoprecipitation mass spectrometry and 143 CTSD interacting proteins were inserted into it. The results of functional cluster analysis showed that 70 CTSD interacting proteins were clustered into 12 clusters according to their functions. Transmembrane transport, cytoskeleton, oxidative phosphorylation, protein synthesis, apoptosis, signal transfer, redox enzyme activity, molecular chaperone, Glucose metabolism. Another 73 CTSD interaction proteins were not involved in cluster. 143 CTSD interaction proteins were analyzed by go. The results showed that the biological process of CTSD interaction proteins was mainly involved in stress response and negative regulation. The analysis of metabolic process, transport process, protein localization and transport process showed that the molecular functions of CTSD interaction proteins were mainly related to protein binding, catalytic activity, purine nucleotide binding, nucleotide binding, cytoskeleton, redox enzyme activity. Structural molecular activity analysis showed that CTSD interaction protein subcells were located in membrane, organelle and vesicle. An analysis of the intermediate. 4kEGG and Biocarta signaling pathways of polymer complexes showed that 143 CTSD interacting proteins were involved in two Biocarta signaling pathways, including hypoxia inducible factor-related signaling pathways, telomere, telomerase, cellular aging and 9 KEGG signaling pathways: oxidative phosphorylation. Pyruvate Metabolism, glycolysis signal Pathway, Adhesion Junction, Antigen processing and presentation, Propionic Acid Metabolism, ribosomal signaling Pathway, The protein-protein interaction network analysis of pentose phosphate pathway showed that the interaction group of CTSD with CTSD was composed of 1: EGFR- HSP90A protein. The result of mass spectrometry was proved to be reliable by immunoprecipitation combined with Western blot to display the binding of EGFR- HSP90A to CTSD. Conclusion 1. 143 interacting proteins of CTSD were identified by immunoprecipitation and mass spectrometry in nasopharyngeal carcinoma cells. In order to further study the function of CTSD protein, the bioinformatics analysis showed that EGFR HSP90A protein interacted with CTSD in nasopharyngeal carcinoma cells. It may play an important role in the invasion and metastasis of nasopharyngeal carcinoma. Two of them, EGFR HSP90A, were verified by immunoprecipitation combined with Western blot, which were consistent with the results of mass spectrometry, suggesting the reliability of the results of mass spectrometry.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.63

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