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標題: | 利用細胞萃取物建立戴奧辛檢測系統之研究 Establishment of a cell-free bioassay for dioxin-like compounds detection with cell lysate |
作者: | Ming-Ching Hsu 徐銘璟 |
指導教授: | 李心予(Hsinyu Lee) |
關鍵字: | 戴奧辛,AhR,ARNT,生物檢測,NanoLuc Luciferase, dioxin,AhR,ARNT,bioassay,NanoLuc luciferase, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 戴奧辛與類戴奧辛化合物是由一群含有兩個氧原子連接一對苯環所構成之衍生化合物的統稱,包括多種氯二聯苯戴奧辛、多氯二聯苯呋喃以及共平面多氯聯苯戴奧辛,這群化合物被認為是已知對人類與動物毒性最強的有機化學物質,一般經由燃燒含有氯的廢棄物產生。其具有高度脂溶性,會經由食物鏈等途徑進入生物體內,然因無法分解而會並持續累積在體內,進而影響生育功能、干擾正常內分泌甚至造成致癌。
目前戴奧辛的檢測多使用氣相層析-高解析度質譜儀 (GC/HRMS) 分析成分與定量,然而其儀器與時間成本使大規模的檢測受到限制。隨著戴奧辛在生物體內致毒機制的研究發展,利用生物檢測方法創造便宜、快速的新檢測技術已成為趨勢。在本論文中,我們建立了能大量表現 AhR 和 NanoLuc luciferase 融合蛋白 (AhR-NL) 的AN細胞株,以及能夠大量表現 AhR-NL 和以組胺酸標定之ARNT (ARNT-His) 的ANA 細胞株,並應用此兩株細胞株建立了兩種具有高靈敏度且快速的活體外戴奧辛生物檢驗方法:第一、由於戴奧辛會促進 AhR-NL 的降解,因此可以使用 AN 細胞裂解液冷光訊號的降低程度來偵測戴奧辛的濃度,其反應時間在一小時以內,並且具有能夠長期冷凍保存的潛力。第二、使用帶有抗組氨酸抗體的瓊脂膠體珠 (agarose beads) 或磁珠 (magnetic beads) 抓取 ANA 細胞裂解液中的ARNT-His,形成 ARNT-beads 複合體。戴奧辛會促進 AN裂解液中的 AhR-NL 與 ARNT-beads 複合體結合,進而使冷光訊號上升,依此來偵測戴奧辛濃度,其所有反應時間也可在一小時內完成。 相較於其他的戴奧辛生物檢測方式,我們在本論文中建立了一套能在活體外進行,且反應時間快速的戴奧辛生物檢測方法,未來可應用於大規模的檢測食物與環境污染物,為人類的健康作出貢獻。 Dioxin and dioxin-like compounds comprise a group of chemicals that contain a double benzene ring-like structure, and are commonly regarded as the most toxic chemical compounds to human and animals. Burning wastes containing chloride commonly produces these compounds. Due to the high hydrophobic quality, accumulation of dioxin in human tissues poses a potential threat to human health. Currently, analytical chemical methods still dominate dioxin detection procedures. However, with gradually increasing knowledge of the dioxin-mediated signal transduction mechanisms, using bioassays to create economic and efficient dioxin detection protocols has become a trend. In this study, we generated AN cell line, which can overexpress AhR-NanoLuc luciferase fusion protein (AhR-NL), and ANA cell line, which can overexpress AhR-NL and histidine-tagged ARNT (ARNT-His), to develop two types of cell-free dioxin bioassays. We found that dioxin induces degradation of AhR-NL, so we can detect a concentration of dioxin by luminescence reduction of AN lysate. The reaction time is within 1 hour, and the cell lysate can still function several months after -80℃ conservation. Additionally, we used anti-His agarose beads or magnetic beads to bind ARNT-His in ANA lysate, forming ARNT-beads complexes. Upon AN lysate reacts with the ARNT-beads complex, dioxin induces the ARNT-beads complex to pull down AhR-NL, which increases luminescence for dioxin detection. The total reaction time is also within 1 hour. Compared to other dioxin bioassays, the cell-free dioxin detection methods we developed in this study are both faster and more sensitive. In the future, we may apply these techniques to detect food and environmental contaminants, which may contribute for human health. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18775 |
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顯示於系所單位: | 生命科學系 |
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