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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 謝淑貞(Shu-Chen Hsieh) | |
dc.contributor.author | Yu-Ting Hsu | en |
dc.contributor.author | 許渝婷 | zh_TW |
dc.date.accessioned | 2021-06-16T05:21:01Z | - |
dc.date.available | 2019-09-10 | |
dc.date.copyright | 2014-09-10 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-15 | |
dc.identifier.citation | 游雅茹 (2012),利用細胞模式評估Bracteanolide A的抗氧化生物功能,國立台灣大學食品科技研究所碩士論文
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56263 | - |
dc.description.abstract | 自由基 (Free radicals) 在細胞中可擔任訊息傳遞介質以維持細胞的正常運作,但是過高的自由基會導致氧化壓力產生,而許多疾病的進程已被證實與氧化壓力有相關聯性,因此找尋潛在抗氧化能力的食材是目前重要的研究課題。抗氧化活性評估方法,不外乎利用體外試管實驗,例如清除DPPH (1,1-diphenyl-2-pricrylhydrazyl)自由基能力、還原力測定等,但是這些分析方法僅是針對消除某幾種特定自由基所設計,且為單純化學反應的評估,無法正確反應生物體的狀況;若以動物體為模式,則消耗時間、金錢、人力且違反3R (Replace, Reduce, Refine) 原則,因此發展一個可快速大量篩檢且即時偵測抗氧化能力的細胞平台是必要的。首先,利用生物技術方法建立一個含有抗氧化反應 (Antioxidant responsive element, ARE) 與分泌型報導基因 (Secretory luciferase reporter gene) 的質體,運用PCR與DNA定序等方式確認其質體的建立後,將其轉殖入人類舌癌細胞Ca9-22中,以抗生素400 μg/mL Neomycin篩選成功轉殖的細胞株。當待測物誘發轉錄因子--NF-E2相關因子二 (Nuclear factor-erythroid 2-related factor-2) 轉運至核內並結合到ARE片段上,將會啟動下游報導基因的轉錄及轉譯,藉由收集培養液即時偵測Nano-Glo Luciferase冷光蛋白的表現量,以代表待測物能誘發抗氧化的能力。本研究已成功建立此細胞平台,並已確認其穩定度與靈敏度,將來可以利用此細胞平台作為抗氧化潛力食材因子的篩選工具。 | zh_TW |
dc.description.abstract | Reactive oxygen species (ROS) act as signaling intermediates for many normal cellular processes, but elevated ROS has been linked to over 150 diseases, including atherosclerosis, diabetes, cancer, and neurodegenerative diseases. These free radicals generated from living body or environment are sources of oxidative stress. Severe oxidative stress leads to DNA damage or/and cell death. Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a key transcription factor that plays a central role in cellular defense against oxidative and electrophilic damages by induction of anti-oxidative enzymes, including heme-oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO1). The objective is to establish a cell platform to screen food factors with potential anti-oxidative function. Our strategy is to construct a plasmid that contains antioxidant responsive element (ARE) driven promoter and secretory form of luciferase reporter genes, and followed by transfecting the plasmid to oral cancer cell line Ca9-22. We selected stable lines with the reporter plasmid insertion in chromosome and evaluate the efficacy of the cell platform by Bracteanolide A, a natural compound that we have previously identified as a strong antioxidant, which could prevent cell from oxidative stress and strongly drive Nrf2 and downstream target genes. We have successfully constructed this cell platform and confirm its stability and sensitivity. In the future, it could be a good tool toward screening food factors with potential antioxidant acitivities. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:21:01Z (GMT). No. of bitstreams: 1 ntu-103-R01641011-1.pdf: 3135043 bytes, checksum: 1c6edfa543fe0e0368549224fe955c80 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員會審定書………………………………………………………………………i
謝誌……………………………………………………………………………………...ii 中文摘要 iv Abstract v 目錄 vi 表目錄 xi 圖目錄 xii 第一章 文獻回顧 1 第一節 氧化壓力傷害與疾病 1 (一)、內生性氧化壓力 2 (二)、外生性氧化壓力 2 (三)、氧化壓力與發炎反應 3 (四)、氧化壓力、疾病與癌症 4 第二節 氧化反應與自由基特性 5 (一)、自由基與活性分子 5 1. 超氧陰離子自由基 (Superoxide anion, O2•−) 6 2. 過氧化氫 (Hydrogen peroxide, H2O2) 6 3. 羥基自由基 (Hydroxyl radical, •OH) 7 4. 單態氧 (Singlet oxygen, 1O2) 7 (二)、自由基與氧化傷害 8 1. 脂質之氧化傷害 8 2. 蛋白質之氧化傷害 9 3. 核酸之氧化傷害 10 (三)、自由基訊息傳遞 10 第三節 抗氧化反應機制 14 (一)、抗氧化物作用原理與機制 14 1. 自由基終止劑 (Free radical terminator) 14 2. 還原劑或氧清除劑 (Reductants and oxygen scavengers) 14 3. 金屬螯合劑 (Metal chelator) 15 (二)、抗氧化系統 15 1. 預防性抗氧化劑 (Preventive antioxidants) / 酵素性抗氧化系統 (Enzymatic antioxidant system) 15 2. 清除自由基抗氧化劑 (Radical scavenging antioxidants) / 非酵素性抗氧化系統 (Non-enzymatic antioxidant system) 16 3. 修復/重新製造之抗氧化劑 (Repair/De novo antioxidants) 17 (三)、抗氧化反應元素(Antioxidant responsive element, ARE) 19 (四)、NF-E2相關因子二 (Nuclear factor-erythroid 2-related factor-2) 及活化機制 20 (五)、NF-E2相關因子二之癌症預防作用 22 第四節 現有抗氧化活性測定方法 23 (一)、抗氧化體外 (In vitro) 試管實驗 23 (二)、抗氧化體內 (In vivo) 實驗 25 第五節 報導基因方法學 28 (一)、螢火蟲冷光酶 (Firefly luciferase, luc) 28 (二)、珊瑚蟲冷光酶 (Renilla luciferase, Rluc) 29 (三)、刺蝦冷光酶 (Nano-Glo luciferase, Nluc) 29 第二章 研究目的與實驗架構 31 第一節 研究目的 31 第二節 實驗架構 32 第三章 材料與方法 33 第一節 實驗試劑與設備 33 第二節 實驗方法 36 (一)、質體製備 36 (二)、質體構築 42 (三)、細胞培養 43 (四)、Secretory Nano-Glo luciferase assay 46 (五)、即時半定量mRNA表現 (Real-time semi-quantification PCR) 48 第三節 統計分析方法 50 第四章 實驗結果 51 第一節 建構即時偵測抗氧化質體 51 1.建構pGL4.18 ARE9-pNL1.3-Nano-Glo luciferase質體 51 2.以限制酶分別處理pGL4.18-ARE9與pNL1.3-Nano-Glo luciferase質體 52 3.以接合酶將pGL4.18-ARE9與pNL1.3-Nano-Glo luciferase質體片斷黏合 54 4. 以限制酶剪切方式確認總質體大小 55 5. 以廠商定序和NCBI BLAST再次確認質體正確性 56 第二節 建立可篩選抗氧化能力食材之細胞平台 58 1. Ca9-22細胞轉殖效率測試 58 2.Neomycin抗生素對於Ca9-22細胞毒殺性測試 62 3.不同劑量t-BHQ誘導抗氧化途徑反應分析 64 4.成功挑選於誘導抗氧化路徑可表現報導質體發冷光的細胞殖株 66 5.於誘導抗氧化途徑下,Ca9-22 clone細胞抗氧化相關基因mRNA表現量 68 6.以細胞免疫螢光染色法確認Ca9-22 clone細胞Nrf2抗氧化路徑可被誘導活化並轉運入細胞核 69 第三節 建立即時偵測抗氧化能力食材細胞平台之測定方式 72 1.負控制組之背景值測定 72 2.抗氧化路徑誘導下,比較報導基因冷光反應與抗氧化相關基因表現量於不同時間點被啟動的情形 73 3.建立全盤掃描式偵測方法 75 4.建立測定標準步驟與校正方法 76 第四節 即時偵測抗氧化能力之細胞平台穩定性測定 77 1.Ca9-22 clone細胞不同代數之穩定度 77 2.Ca9-22 clone細胞反覆冷凍-解凍特性 79 第五節 藉由即時偵測抗氧化能力之細胞平台篩選具抗氧化潛力食材因子 81 第五章 討論 83 第六章 結論 86 第七章 參考文獻 87 | |
dc.language.iso | zh-TW | |
dc.title | 以Ca9-22建立可篩選具抗氧化潛力之食材的細胞平台 | zh_TW |
dc.title | Establishment and Evaluation of Ca9-22
Cell Platform for Screening Food Factors with Anti-oxidative Potential | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 許順堯(Shun-Yao Hsu) | |
dc.contributor.oralexamcommittee | 黃智興(Tze-Sing Huang),郭靜娟,羅翊禎 | |
dc.subject.keyword | 抗氧化,細胞平台,分泌型報導基因 (Secretory luciferase reporter gene),抗氧化反應片段(Antioxidant responsive element, ARE),NF-E2相關因子二 (Nuclear factor-erythroid 2-related factor-2),Ca9-22, | zh_TW |
dc.subject.keyword | Antioxidant,Cell platform,Secretory luciferase reporter gene,ARE,Nrf2,Ca9-22, | en |
dc.relation.page | 96 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-16 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 食品科技研究所 | zh_TW |
顯示於系所單位: | 食品科技研究所 |
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