化學酵素冷光基因表現法應用於環境基質之研究

Application of Bioassay on Enviromental Samples

游彥勝(Yen-Sheng You)

指導教授 ： 王雅玢

中原大學/工學院/土木工程研究所/碩士(2010年)

https://doi.org/10.6840/cycu201000794

摘要

中文摘要
戴奧辛與呋喃為環境中持久性的有機污染物，具有生物蓄積性與親脂性，不易被代謝，其中毒性最強之2,3,7,8-TCDD被國際癌症組織公告為確定人類致癌物質，對人體中的肝臟、皮膚、生殖系統及免疫系統具有毒害性而備受矚目。
過去分析戴奧辛及其類化合物以化學分析HRGC/HRMS為主，但是化學分析分析樣品費時、分析費用高、無法短時間處理大量樣品，因此生物分析的發展可彌補化學分析之不足，國外商業化細胞株因為專利費用高，推廣不易，本研究希望能建立本土性生物快速篩選法(Huh7-DRE-Luc細胞株)的標準操作程序，並先利用液/固態標準品進行參數測試，最後進行環境中底泥、土壤、煙道氣真實樣品的實測分析，進行化學測值與生物測值之比對，找出生物分析之適用性。
研究結果顯示，Huh7-DRE-Luc細胞株對於固態與液態標準品測試結果皆呈現良好之穩定結果，生物分析測值比上化學分析測值(BIO/CHE)平均值分別為5.48與3.98，且當TCDD濃度大於78.7 pM時，其劑量反應曲線才具有較高的準確性；對環境基質之測試顯示，底泥與土壤樣品具有相同趨勢，在高濃度樣品區間，BIO/CHE比值較低(2.0~7.5)，在低濃度樣品區間，BIO/CHE比值較高(37.3與42.3)，而對煙道氣樣品而言，冷光值達20000 RLU以上時，BIO的預測值才會比較接近CHE的測值，研究初步結果顯示此細胞株目前對濃度高的樣品具有較穩定的檢測效果。

關鍵字

環境基質 ； 冷光分析法 ； 生物快速篩檢法 ； 戴奧辛/呋喃

並列摘要

Abstract
Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are polychlorinated aromatic compounds that are accumulated mainly in fatty tissues due to their high lipophilicity, long biological half-lives, and high resistance to biodegradation. 2,3,7,8 - TCDD, the most toxic PCDD/Fs, was classified as carcinogenic to humans (Group 1). General population exposure to PCDD/DFs can induce several adverse health effects including reproductive, hormonal, immunological, and carcinogenic effects .
High-resolution gas chromatography / high-resolution mass spectrometry (HRGC/HRMS) is a certified method to determine dioxin concentrations in Taiwan. However, it costs a lot and takes a lot of time. The chemical activated luciferase expression bioassay has been recently established and showed potential to analyze PCDD/Fs. The bioassay is an in vitro luciferase-reporter gene assay for detecting the TEQ levels of dioxins and dioxin-like compounds based on their high affinity with aryl hydrocarbon receptor (AhR). Compared with HRGC/HRMS, the bioassay is a rapid and low-cost screening method particularly for fast-screen identification of highly dioxin-contaminated samples from environment and food. However, the commercial cell-line is too expensive and hard to be popularized to industry. In this study, a stably recombinant cell-line, Huh7-DRE-Luc cell, was established. The capability of Huh7-DRE-Luc cell (DRE-Luciferase assay) was tested by 2,3,7,8-TCDD standard, CRM powder, sediments, soils and flue gas samples.
The results indicated that the ratios of DRE-Luciferase assay (BIO) to HRGC/HRMS (CHE) for solid and liquid standards were 5.48 and 3.98, respectively. When 2,3,7,8-TCDD concentration was greater than 78.7 pM, the reliability of dose-response curve increased. For soil and sediment samples, the ratio of BIO/CHE ranged from 2.0 to 7.5 among high concentration samples. The ratio of BIO/CHE ranged from 37.3 to 42.3 among low concentration samples. For flue gas samples, when the relative luciferase activity (RLU) was greater than 20000, the result of DRE-Luciferase assay was very close to that of HRGC/HRMS. Based on the preliminary results, the Huh7-DRE-Luc cell is suggested to be qualified for analysis of high concentration samples.

並列關鍵字

DRE-Luciferase assay ； PCDD/Fs ； environmental matrice ； RLU.

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