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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 李心予(Hsinyu Lee) | |
| dc.contributor.author | Chun-Hung Hsieh | en |
| dc.contributor.author | 謝俊弘 | zh_TW |
| dc.date.accessioned | 2021-06-13T15:46:27Z | - |
| dc.date.available | 2013-07-07 | |
| dc.date.copyright | 2008-07-07 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-06-30 | |
| dc.identifier.citation | 1 Abbott, B. D., Schmid, J. E., Brown, J. G., Wood, C. R., White, R. D., Buckalew, A. R., and Held, G. A. (1999). RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone. Toxicological Sciences. 47 (1), 76-85.
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Improvement of chemically-activated luciferase gene expression bioassay for detection of dioxin-like chemicals. Biomedical and Environmental Sciences. 15 (1), 58-66. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37836 | - |
| dc.description.abstract | 戴奧辛（dioxin）是一群含多氯聯苯化合物之總稱，由於具有高度脂溶性，多經由食物鏈積存於生物體內，影響賀爾蒙之調控，甚至造成細胞癌化，其中2,3,7,8-TCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin)是目前所知毒性最強之污染物。目前我國檢測戴奧辛大多使用氣相層析-高解析度質譜儀分析(GC/HRMS)等化學方法，然而隨著戴奧辛在生物體內毒理機制了解與日俱增，先後有ELISA、CALUX等生物檢測戴奧辛方法，由於具備快速、便宜、操作簡單等優勢，目前應用生物檢測方法來篩檢戴奧辛樣品逐漸成為主流。因此，本研究根據細胞辨識戴奧辛等環境污染物反應，分別以小鼠戴奧辛受器mAHR (mouse aryl hydrocarbon receptor)-小鼠熱休克蛋白90 mHSP90 (mouse heat shock protein 90)組合與人類戴奧辛受器hAHR (human aryl hydrocarbon receptor)-人類戴奧辛受器核轉置蛋白hARNT(human AHR nuclear translocator)組合，藉由螢光共振能量轉移技術(Fluorescence Resonance Energy Transfer，FRET)訊號偵測戴奧辛污染程度，在時間及成本上占有相對優勢，且較能真實反應環境汙染因子對生物體的毒害程度。實驗設計上，分別建構mAHR-CFP與mHSP90-YFP以及hAHR-CFP與hARNT-YFP的表達質體，送入大鼠肝癌H4IIEC3細胞中，結果發現螢光共振能量轉移訊號與戴奧辛污染程度有依賴關係，在mAHR-CFP與mHSP90-YFP組合部分，隨著不同劑量之戴奧辛而訊號下降；在hAHR-CFP與hARNT-YFP組合部分，則隨著不同劑量而訊號上昇。根據研究結果，未來具有作為輔助戴奧辛等環境污染物大量篩選檢測工具之可行性。 | zh_TW |
| dc.description.abstract | Dioxins comprise a group of compounds which contain a double aromatic ring-like structure. They are among the most prevalent and toxic environmental pollutants. Accumulation of dioxins in human tissues poses a potential threat to human health. Currently, analytical chemical procedures dominate dioxin-detection protocols. In this study, we established 2 set of fluorescence resonance energy transfer (FRET)-based dioxin-detection bioassays. We generated plasmids encoding fusion proteins of mAHR (Mouse aryl hydrocarbon receptor) and mHSP90 (Mouse heat shock protein 90) with CFP and YFP , and the other set by the same method, We generated plasmids encoding fusion proteins of hAHR (Human aryl hydrocarbon receptor) and hARNT (Human AHR nuclear translocator) with CFP and YFP, respectively. Constructs were transiently co-transfected into rat hepatoma cell line, H4IIEC3 cells. Our results showed that FRET signals were detected in mAHR-CFP and mHSP90-YFP co-transfected H4IIEC3 cells. Dioxin treatments down-regulated FRET signals in these transfected cells in a dose-dependent manner. On the other hand, no FRET signals were detected in hAHR-CFP and hARNT-YFP-transfected H4IIEC3 cells. In contrast, dioxin treatments up-regulated FRET signals in these transfected cells in a dose-dependent manner. This work highlighted the potential of using FRET technique in the detection of dioxin-like compounds. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T15:46:27Z (GMT). No. of bitstreams: 1 ntu-97-R95b41004-1.pdf: 1588409 bytes, checksum: 81b9984355c9233da499a944f8b30247 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………..…………...… I
誌謝……………………………………………………………………......…….. II 中文摘要……………………………………………………………………….... III Abstract…………………………………………………………………………….. IV 第一章 前言……………………………………………………...……………… 1 1.1 戴奧辛簡介………………………………………..……………………. 1 1.2 戴奧辛於細胞的作用機制………………………..……………………. 5 1.3 戴奧辛的檢測方法…………………………………..…………………. 7 1.4螢光共振能量轉移 (FRET)…………………………….……………… 10 第二章 材料與方法………………………………………………………..……. 14 2. 1 試劑…………………………………………………………….…….…. 14 2. 2 細胞培養…………………………………………………….………….. 14 2. 3 質體建構…………………………………………………….………….. 14 2. 4 細胞轉殖………………………………………………….…………….. 16 2. 5 細胞株選殖……………………………………………….…………….. 16 2. 6 戴奧辛配製…………………………………………….……………….. 16 2. 7 螢光共振能量轉移量化………………………………….…………….. 16 2.8 共免疫沉澱法與西方墨點法分析…………………………………….. 17 2.9 建立基因轉殖雞……………………………………………………….. 18 2. 10 統計分析…………………………………………………………….. 18 第三章 實驗設計……………………………………………………………….. 19 3. 1戴奧辛in vivo生物檢測系統之建立………………………………….. 19 3.2 戴奧辛 in vitro生物檢測系統之建立………………………………… 27 第四章 結果…………………………………………………………………….. 29 第五章 討論…………………………………………………………………….. 34 第六章 附圖…………………………………………………………………….. 41 圖一. 戴奧辛結構及2,3,7,8-TCDD……………………………………....... 41 圖二. 螢光共振能量轉移原理……...………………………………............ 42 圖三. 以AHR與HSP90蛋白分子作為戴奧辛生物檢測系統 實驗原理闡述……………………………………………………….. 43 圖四. mAHR與mHSP90戴奧辛生物檢測系統之實驗設計……………... 44 圖五. 建構mAHR-CFP與mHSP90-YFP質體…………………………... 45 圖六. 戴奧辛促使mAHR轉進核內現象…………………………………. 46 圖七. 戴奧辛誘導mAHR-CFP與mHSP90-YFP螢光共振能量轉移 訊號強度下降…………………………………………….…………. 47 圖八. 戴奧辛誘導mAHR-CFP與mHSP90-YFP螢光共振能量轉移 訊號強度下降有劑量依賴的關係………………………….……….. 48 圖九. 戴奧辛誘導mAHR-CFP與mHSP90-YFP螢光共振能量轉移 訊號強度下降有時間依賴的關係…………………………….…..… 49 圖十. 以AHR與ARNT蛋白分子作為戴奧辛生物檢測系統 實驗原理闡述……………………………………………………….. 50 圖十一. hAHR與hARNT建構之生物檢測系統實驗設計………….....…. 51 圖十二. 建構hAHR-CFP與hARNT-YFP質體…………………………... 52 圖十三. 戴奧辛誘導hAHR-CFP與hARNT-YFP螢光共振能量轉移 訊號上昇且促使hAHR-CFP轉進核內現象…………….…...…. 53 圖十四. 戴奧辛誘導hAHR-CFP與hARNT-YFP螢光共振能量轉移 訊號強度上昇有劑量依賴的關係…….……………….……...…. 54 圖十五. 以螢光共振能量轉移技術作為戴奧辛生物檢測模式之結論...… 55 圖十六. 挑選hAHR-CFP與hARNT-YFP穩定細胞株………………....... 56 圖十七. AHR-HSP90蛋白分子於in vitro相互作用之實驗設計…………. 57圖十八. 應用共免疫沉澱實驗探討AHR-HSP90於in vitro相互作用 的可行性…………………………………………………..…..…... 58 圖十九. 建立hAHR/hARNT基因轉殖雞………………………….……… 59 第七章 附表……………………………………………………………………... 60 附表一. 戴奧辛毒性當量因子………………………………………..…….. 60 附表二. 戴奧辛生物檢測模式比較分析……………………………..…….. 61 附表三. 螢光蛋白分子光譜特性……………………………………..…….. 62 參考文獻…………………………………………………………………….…….. 63 | |
| dc.language.iso | zh-TW | |
| dc.subject | 戴奧辛 | zh_TW |
| dc.subject | 螢光共振能量轉移 | zh_TW |
| dc.subject | 戴奧辛生物檢測方法 | zh_TW |
| dc.subject | Dioxin | en |
| dc.subject | Fluorescence resonance energy transfer | en |
| dc.subject | Dioxin bioassay | en |
| dc.title | 以螢光共振能量轉移技術作為戴奧辛生物檢測模式之研究 | zh_TW |
| dc.title | Establishment of a Fluorescence Resonance Energy Transfer-based bioassay for detecting dioxin-like compounds | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 潘建源(Chien-Yuan Pan),張簡國平(Gou-Ping Chang-Chien),張文興(Wen-Hsing Chang) | |
| dc.subject.keyword | 戴奧辛,螢光共振能量轉移,戴奧辛生物檢測方法, | zh_TW |
| dc.subject.keyword | Dioxin,Fluorescence resonance energy transfer,Dioxin bioassay, | en |
| dc.relation.page | 71 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-07-01 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究所 | zh_TW |
| Appears in Collections: | 動物學研究所 | |
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| File | Size | Format | |
|---|---|---|---|
| ntu-97-1.pdf Restricted Access | 1.55 MB | Adobe PDF |
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