以Ca9-22建立可篩選具抗氧化潛力之食材的細胞平台

Establishment and Evaluation of Ca9-22 Cell Platform for Screening Food Factors with Anti-oxidative Potential

許渝婷(Yu-Ting Hsu)

指導教授 ： 謝淑貞

共同指導教授 ： 許順堯(Shun-Yao Hsu)

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.01367

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

Antioxidant ； Cell platform ； Secretory luciferase reporter gene ； ARE ； Nrf2 ； Ca9-22

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