2013-08-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/662436摘要：戴奧辛包含多種對環境有害的有毒化合物，這些化合物經由芳香烴受器所調控的傳訊路徑影響細胞的行為。近年來，研究發現一些內生性的代謝物能夠與芳香烴受器結合並影響細胞的生理反應，然而其他內生性芳香烴受器之受質仍有待發現。藉由生物冷光能量共振轉移之技術，本實驗室開發出相當靈敏的檢測戴奧辛化合物方法，在人類胚胎腎臟細胞株T-REx 293中表現與芳香烴受器相接之水母冷光&#37238;，以及與熱休克蛋白90或芳香烴受器核轉移蛋白相接之黃色螢光蛋白，不同濃度之戴奧辛化合物將引發不同強度之螢光訊號，且此螢光強度和戴奧辛化合物之濃度呈現正比關係。由於此方法的偵測極限可達10-16莫耳濃度，如此靈敏的偵測系統可作為尋找內生性芳香烴受器之受質的有效方法。本計畫利用不同成長階段之斑馬魚胚胎作為材料，適當分層萃取純化後，利用前述偵測戴奧辛的方法尋找可能的目標受質，最後利用液相層析質譜技術確定此化合物之結構，此內生性化合物將可供未來進行更深入研究。<br> Abstract: Dioxins comprise a group of toxic compounds, which are among the most prevalent and toxic environmental pollutants. These compounds affect cellular behaviors through activating the AHR-mediated signaling pathway. Recently, several endogenous metabolites were identified unintentionally to be able to bind to AHR and suggested their roles as physiological regulators. However, the identities of other endogenous AHR ligands remain elusive. In our laboratory, we have developed an extremely sensitive BRET based assay for dioxin like compounds. AHR fused renilla luciferase (RL), with either HSP90 or ARNT fused yellow fluorescent protein (YFP) constructs were stably co-transfected into human embryonic kidney cell line, T-REx 293 cells. In the presence of different concentrations of dioxin like compounds, the fluorescent signals generated from YFP activated by light emitted from RL will varied in a dose-dependent manner. Moreover, the detection limits are as low as 10-16 M. This extremely sensitive system suggests the potential to identify endogenous AHR ligands. In this proposal, we attempt to isolate and fractionate large amount of zebrafish embryos from different developmental stages. The purify metabolites will be subjects to the dioxin assays developed. The positive fractions will be further fractionated and the ultimate identity of the isolated compounds will be delineated using LC-MS. These procedures should decipher the endogenous dioxin-like compounds and their physiological roles could be explored in the future.