建立以生物反應為基礎的戴奧辛檢測方法

The luciferase recombinant cell culture bioassay system for detecting dioxin

黃怡瑋(Yi-Wei Huang)

指導教授 ： 李心予

國立臺灣大學/生命科學院/動物學研究研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00157

摘要

戴奧辛（Dioxin）是一種苯環類化合物之統稱，也是一種著名的環境毒物，由於具有高度脂溶性，故常經由食物鏈途徑在生物體內積存，對其生理系統會造成很大的影響。目前國內外針對戴奧辛的檢測方法仍是以化學分析方法為主流，費時且耗費昂貴，有鑒於戴奧辛生物檢測對戴奧辛生物危害的直接意義，本研究期望可以利用細胞反應來辨識戴奧辛環境污染物，並建立一套更方便的生物檢測系統。以及高靈敏度的定性分析優勢，建立戴奧辛生物檢測技術。
根據目前的研究，已經知道當戴奧辛進入細胞後會和帶有兩個Heat Shock Protein 90(HSP90)的芳香族碳氫化合物受器(Aromatic hydrocarbon receptor)結合而形成複合物，送入細胞核之後此複合物會分離，藉由一連串下游分子之作用後輾轉傳遞至DNA上和戴奧辛識別單元(dioxin response elements)結合，使得受其調控的基因大量表現，進而影響整個生物體的生理系統。針對此傳遞路徑的特色，我們建立兩個含有戴奧辛識別單元和螢光基因(luciferase gene)的表達質體，分別送入不同的細胞株中，藉由螢光的定量做出一組標準曲線圖，使其成為偵測環境污染物的工具。
目前的實驗結果發現，RAW264.7細胞中戴奧辛量及螢光表現並沒有很好的線性關係，不適合拿來做生物檢測系統，而針對H4IIEC3和Hepa1C1C7細胞，我們已經建立戴奧辛濃度-螢光表現標準曲線圖，當有未知濃度的檢體時，便可就由和標準曲線螢光量的比對來得知戴奧辛的含量，這種以生物反應為基礎的戴奧辛檢測方法除了快速、方便、成本低廉，而且直接偵測戴奧辛的毒性總量，將比傳統化學分析方法更適合用作日常例行性檢驗的戴奧辛監控。

關鍵字

生物檢測 ； 戴奧辛 ； 環境污染

並列摘要

Dioxin which largely contains aromatic compounds, also known as lipophilic compounds, is among the most widespread environmental pollutants. Accumulation of the compounds in human tissue poses a potential threat to human health. Currently, analytical chemical methods still dominate the mainstream dioxin detection. However, the methods are not only costly but also time-consuming, which often precludes their use in routine monitoring. Assays employing bioassay offer a powerful alternative to chemical analyses. The methods based on biological effects are ‘toxicological specificity’, which refers to the relationship between the assay response and the toxic potentiality rather than simply the contaminant concentrations of the sample being analyzed. This research uses the cell response to identify dioxin contanimants, expecting to establish a highly sensitive dioxin detecting technique.
Surveying existent studies, we have concluded that dioxin can combine with the aromatic hydrocarbon receptor (AHR) and heat shock protein 90 (Hsp90) complex in cytoplasm. This complex will separate in cell nucleus, through a series of signal transmitting and then bind itself to dioxin response elements to effect gene expression. With this characteristic, two reporter vectors can be constructed . The reporter vectors which contain the luciferase gene and dioxin response elements are then transfected to different cell lines. When used together with a sensitive luminometer, the cells express different light intensity depending on their dioxin concentrations.
By the examination of our data, it can be observed that the fine light intensity-dioxin concentration curves appear in H4IIEC3 and Hepa1C1C7, but not in RAW264.7. As a result, in comparison with the curves, the dioxin concentration of an unknown sample can be easily detected. The major advantages of such biological, mechanism-based methods are their toxicological specificity, rapidity, and low costs. So the use of this cell culture bioassay will be more convenient and efficient in routine monitoring.

並列關鍵字

dioxin ； bioassay

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