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研究生：	廖猛崴 Liao, Mong-Wei
論文名稱：	應用報導基因檢測法探討臺灣河川水及懸浮固體中類戴奧辛物質之分布 Application of a reporter gene assay to investigate the distribution of dioxin-like compounds in dissolved phase and suspended solids from Taiwanese rivers
指導教授：	周佩欣 Chou, Pei-Hsin
學位類別：	碩士 Master
系所名稱：	工學院 - 環境工程學系 Department of Environmental Engineering
論文出版年：	2011
畢業學年度：	99
語文別：	中文
論文頁數：	120
中文關鍵詞：	報導基因檢測法、類戴奧辛物質、懸浮固體物、河川水體
外文關鍵詞：	Dioxin-like compounds, Reporter gene assay, Suspended solids, River water
相關次數：	點閱：141 下載：13
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　　本研究利用基因重組酵母菌對台灣河川中的類戴奧辛物質進行檢測，並嘗試以高效率液相層析儀將樣本複雜度降低後再作進一步分析，期望藉由此種方式找出河川中主要貢獻類戴奧辛活性的物質，並探討類戴奧辛活性較高的河川其水相與懸浮固相所含的類戴奧辛物質是否具有相關性，研究中也額外使用rec-assay針對某些特殊樣本進行基因毒性鑑定。
　　實驗結果顯示不論在水相或懸浮固相中，鹽水溪、阿公店溪及二仁溪流域皆具有相當高的類戴奧辛活性，其中又以二仁溪的類戴奧辛當量濃度為最高，於水相樣本中測得7.36 ± 0.70 nM β-NF-EQ，懸浮固體樣本中測得397.8 ± 38.1 μg β-NF/g SS-EQ。而水相樣本在高濃縮倍率或環境濃度10倍的情況下，大部分具有高類戴奧辛活性的樣本，均會造成菌體生長抑制。這些樣本經過液相層析儀分離之後發現極為可能是相同的生長抑制物質，隨後以rec-assay檢測發現均具有基因毒性，然而這些物質在具有高類戴奧辛活性的懸浮固體樣本中並未被發現。經由層析圖譜可知，兩相之間導致類戴奧辛活性的物質並不相同，而存在於二仁溪、鹽水溪及阿公店溪水相中導致類戴奧辛活性之物質可能源自於工業廢水。本研究中應證基因重組酵母菌能快速且有效反應河川流域中類戴奧辛活性，可做為類戴奧辛污染之快速篩檢工具。

　　This study used a recombinant yeast assay and high performance liquid chromatography (HPLC) fractionation process to separate and detect the dioxin-like compounds in dissolved phase and suspended solids from Taiwanese rivers. HPLC fractionation process was used to fractionate samples and further bioassay analysis was undertaken to investigate the dioxin-like activity of fractions. The aims of this study were to isolate the major compounds that contributed to high dioxin-like activity, and to further investigate the distribution of dioxin-like compounds in dissolved phase and suspended solids. Additionally, genotoxicity of some specific fractions was also evaluated using the rec assay.
　　For most of the tested samples, higher dioxin-like activity was detected in Erren, Agongdian, and Yanshuei rivers. The highest equivalent concentration of dioxin-like compounds was 7.36 ± 0.70 nM β-NF-EQ in dissolved phase and 397.8 ± 38.1 μg β-NF/g SS-EQ in suspended solids at Erren river basin. According to bioassay results and HPLC chromatograms, similar substances which caused dioxin-like activity and yeast growth inhibition may exist in 21min fractions of Erren, Agongdian and Yanshuei rivers, while the composition of dioxin-like compounds were different in dissolved phase and suspended solids. Genotoxic effect was also detected in the fraction of these samples, suggesting that the compounds inducing dioxin-like activity may also cause genotoxicity. The compounds which caused dioxin-like activity from Erren, Yanshuei, and Agongdian probably derived from industrial wastewater. The recombinant yeast assay described in this study can be useful to rapidly and efficiently evaluate dioxin-like activity for screening river basin pollution.

摘  要 .................................... I 
Abstract...................................II 
誌謝 ......................................IV 
目錄 ......................................VI 
圖目錄 ....................................X 
表目錄 ....................................XIII 
第一章 前言 ...............................1 
1-1 研究緣起 ..............................1 
1-2 研究目的 ..............................2 
第二章 文獻回顧 ...........................3 
2-1 內分泌干擾物質 ........................3 
2-1-1 多環芳香烴受體 ......................4 
2-1-2 戴奧辛物質 ..........................5 
2-1-3 類戴奧辛物質定義 ....................7 
2-1-4 目前已知的類戴奧辛物質及來源 ........7 
2-2 生物檢測方法 ..........................9 
2-2-1 報導基因檢測方法 ....................10 
2-2-2 EROD檢測方法 ........................13 
2-2-3 酵素免疫分析法 ......................15 
2-2-4 生物活體檢測 ........................17 
2-3 化學分析方法 ..........................18 
2-3-1 氣相層析系統於分析有機物上的應用 ....18 
2-3-2 液相層析系統於分析有機物上的應用 ....19 
2-4 採樣河川流域現況 ......................21 
2-4-1 低污染河川 ..........................22 
2-4-2 中度污染河川 ........................23 
2-4-3 重度污染河川 ........................25 
第三章 實驗步驟與方法 .....................26 
3-1 樣本採集 ..............................26 
3-1-1 水樣採集及懸浮固體取得方式 ..........26 
3-1-2 採集地點及位置 ......................29 
3-2 實驗材料及設備 ........................34 
3-2-1 實驗材料 ............................34 
3-2-2 實驗設備 ............................37 
3-3 實驗流程 ..............................39 
3-4 樣本前處理 ............................40 
3-4-1 水樣前處理 ..........................40 
3-4-2 懸浮固體樣本前處理 ..................41 
3-5 重組基因酵母菌報導基因試驗法 ..........43 
3-5-1 檢測方法原理（YCM3 AhR assay） ......43 
3-5-2 檢測方法實驗流程 ....................44 
3-5-3 類戴奧辛活性的計算 ..................46 
3-6 基因毒性檢測 ..........................48 
3-6-1 檢測方法原理（rec-assay） ...........48 
3-6-2 檢測方法實驗流程 ....................48 
3-7 RP-HPLC樣本分離 .......................51 
3-7-1水相樣本 .............................51 
3-7-2懸浮固體物 ...........................52 
第四章 結果與討論 .........................53 
4-1 河川水相樣本之AhR誘導活性 .............53 
4-1-1 低污染河川 ..........................53 
4-1-2 中度污染河川 ........................56 
4-1-3 重度污染河川 ........................62 
4-1-4 小結（水相樣本） ....................65 
4-2 懸浮固體樣本之AhR誘導活性 .............67 
4-2-1 鹽水溪流域 ..........................67 
4-2-2 二仁溪流域 ..........................68 
4-2-3 阿公店溪流域 ........................72 
4-2-4 高屏溪流域 ..........................73 
4-2-5 小結（懸浮固相樣本） ................75 
4-3 HPLC之分析結果 ........................76 
4-3-1二仁溪樣本 ...........................76 
4-3-2阿公店溪樣本 .........................87 
4-3-3其它流域之樣本 .......................91 
4-4 水相及懸浮固體類戴奧辛之分布 ..........95 
4-5 二仁溪水相樣本基因毒性檢測 ............98 
第五章 結論與建議 .........................102 
5-1 結論 ..................................102 
5-2 建議 ..................................105 
參考文獻 ..................................106 
自述 ......................................120
                                    

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