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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61879完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李心予(Hsinyu Lee) | |
| dc.contributor.author | Tzu-Ching Tsai | en |
| dc.contributor.author | 蔡子晴 | zh_TW |
| dc.date.accessioned | 2021-06-16T13:16:56Z | - |
| dc.date.available | 2018-08-29 | |
| dc.date.copyright | 2013-08-29 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-07-29 | |
| dc.identifier.citation | 5 Reference
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Fletcher, et al., Optimisation of bioluminescent reporters for use with mycobacteria. PLoS ONE, 2010. 5(5): p. e10777. 51. Soshilov, A. and M.S. Denison, Ligand displaces heat shock protein 90 from overlapping binding sites within the aryl hydrocarbon receptor ligand-binding domain. Journal of Biological Chemistry, 2011. 286(40): p. 35275-82. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61879 | - |
| dc.description.abstract | 戴奧辛是一種已知的環境毒性化合物，對人體會造成傷害，其作用於芳香烴受體上時，使原本與芳香烴受體結合的熱蛋白脫離，而與戴奧辛結合的芳香烴受體會從細胞質進入細胞核內，和核內的芳香烴核轉位蛋白結合形成複合體，此複合體會啟動下游基因如 CYP1A1 等等表現，產生毒性影響細胞的生理作用。因此，我們以芳香烴受體的訊息傳遞路徑為基礎，建構出同時大量表現冷光的芳香烴受體以及組胺酸標定的熱蛋白的細胞，並且收集細胞裂解液，與含有抗組胺酸的抗
體的瓊脂凝膠體珠混和，最後，得到帶有芳香烴受體與熱蛋白的複合體。當戴奧辛作用時，帶有冷光的芳香烴受體會與戴奧辛結合，並且離開熱蛋白，造成芳香烴受體被降解，進而使冷光訊號減少。相較於以往的化學檢測和其他的生物性檢測，我們利用穩定的冷光訊號，建立了一個更有效且具有專一性的戴奧辛檢測系統。 | zh_TW |
| dc.description.abstract | Dioxin is a chemical compound that persists in the environment for a long time and is toxic to humans. In the cytoplasm, it binds to the aryl hydrocarbon receptor (AhR)
and causes the receptor to dissociate from the heat shock protein 90 (Hsp90). Furthermore, dioxin-bound AhR translocates into the nucleus and binds to the aryl hydrocarbon receptor nuclear translocator (Arnt), triggering downstream signaling and affecting the physical behavior of cells. Here we propose a dioxin detection system based on the AhR pathway. We generated a cell line that overexpressed nano-luciferase-tagged AhR and His-tagged Hsp90 for the preparation of cell-free extracts. The AhR-Hsp90 complex was pulled down by anti-His beads. On treatment with dioxin, the luminescence signal generated by AhR nano-luciferase decreased. This study outlines a new cell-free system to detect dioxin-like compounds by observing the stability of bioluminescence signals. This efficient system is more sensitive and less time consuming than chemical methods and other cell-based bioassays. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T13:16:56Z (GMT). No. of bitstreams: 1 ntu-102-R00b41023-1.pdf: 1976361 bytes, checksum: c082717f854bbcfedb7a4b8b0c8f1ffe (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書 ................................................... i 致謝............................................................... ii 中文摘要.......................................................... iii Abstract........................................................................................................................ iv 1 Introduction............................................................................................................... 1 1.1 Dioxins and dioxin-like compounds.....................................................................1 1.2 Aryl hydrocarbon receptor (AhR) pathway ........................................................ 1 1.3 Dioxin detection assay ....................................................................................... 2 1.4 Nanoluciferase ...................................................................................................4 2 Materials and Methods............................................................................................. 5 2.1 Plasmid construction............................................................................................ 5 2.2 Production of lentiviral stocks ...........................................................................6 2.3 Generation of stably transfected cell lines ......................................................... 6 2.4 Cell culture .........................................................................................................7 2.5 Preparation of cell-free extracts ........................................................................7 2.6 Western blotting.................................................................................................8 2.7 Pull-down assay .................................................................................................9 2.8 Chemical preparation ........................................................................................ 9 2.9 Detection of dioxin-like compounds by luminescence plate reader ................. 10 2.10 RNA isolation and reverse-transcription (RT) ...............................................10 2.11 Quantitative real-time PCR ............................................................................10 2.12 Statistical analysis ............................................................................................ 11 3 Results...................................................................................................................... 12 3.1 Establishment of a new ANH cell system........................................................... 12 3.2 The construction of plasmids within ANH cells ...............................................12 3.3 Expression levels of Hsp90................................................................................13 3.4 Compare the strength and stability of luciferase signals of AhR-Rluc and AhR-NL in the cell lysate ........................................................................................ 13 3.5 Dioxin detection by ANH degradation ............................................................. 14 3.6 Dioxin detection by ANH pull-down system ..................................................... 15 4 Discussion................................................................................................................. 17 5 Reference................................................................................................................. 20 6 Figures .................................................................................................................... 29 7 Table ....................................................................................................................... 51 | |
| dc.language.iso | zh-TW | |
| dc.title | 建立芳香烴受體和熱反應蛋白複合體之戴奧辛檢測系統 | zh_TW |
| dc.title | Establishment of an Aryl Hydrocarbon Receptor-Heat
Shock Protein 90 (AhR-Hsp90) Pull-down System to Detect Dioxin and Dioxin-like Compounds | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳俊宏(Chen, Jiun-Hong),黃元勵(Yuan-Li Huang),廖永豐(Yung-Feng Liao) | |
| dc.subject.keyword | 戴奧辛,芳香烴受體,生物性檢測,冷光, | zh_TW |
| dc.subject.keyword | Dioxin,AhR,Hsp90,bioassay,nanoluciferase,beads, | en |
| dc.relation.page | 51 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-07-29 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
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