新穎病毒感染肝腫瘤細胞戴奧辛生物篩檢技術之研究

A novel dioxin bioassay with the adenovirus-infected-hepatoma cells for rapid screen of soil samples

陳建宇(Chien-Yu Chen)

指導教授 ： 趙浩然

國立屏東科技大學/工學院/環境工程與科學系所/碩士(2013年)

https://doi.org/10.6346/NPUST.2013.00130

摘要

戴奧辛污染是國人極為關注之環境議題且戴奧辛對於自然環境及人類健康之毒害日益受到重視，戴奧辛造成的毒性反應範圍相當廣泛，可分為皮膚毒性、免疫、神經、內分泌、生殖毒性與致癌性等。一般檢測戴奧辛的方法是利用高解析氣相層析質譜儀(High resolution gas chromatograph/high resolution mass spectrometer, HRGC/HRMS)來進行分析，由於此項化學分析成本過高且耗時，因此需要快速且低成本的分析方法進行大量樣品分析。因此化學活化冷光酵素報導基因法（Chemically Activated LUciferase gene eXpression，CALUX）被廣泛的接受使用於分析各種相關類型的基質，如生物體、環境檢體、食品與飼料樣品中2,3,7,8-TCDD和類戴奧辛化合物。對於大量樣品而言，化學活化冷光酵素報導基因法是一種快速、靈敏與便宜的生物分析測定戴奧辛工具。因此本研究以腺病毒感染鼠類肝腫瘤細胞株(Ad-DRE-Luc/H4IIE)之本土化戴奧辛生物分析方法(Ad-DR bioassay)為研究基礎，嘗試降低Ad-DRE-Luc/H4IIE細胞株於戴奧辛樣品暴露時之溫度，並改以含有較高戴奧辛反應單元之腺病毒進行實驗。利用此方法進行2,3,7,8-TCDD標準品、環境基質樣品(Certified Reference Materials, CRM) (Dioxins and Furans in Reference Sediment DX-1)與土讓樣品測試，相較於先前未降溫與未增加戴奧辛反應單元生物偵測系統，於樣品暴露時降低溫度至33℃並改變戴奧辛反應單元，從原來4倍戴奧辛反應單元提升至6倍戴奧辛反應單元，穩定度有明顯改善。本次研究方法之偵測極限值為3.2 pM，土壤樣品預估值與GC-HRMS兩者數據分別取對數轉換後進行一次線性迴歸，兩者具有良好的關聯性(R2=0.6614)，由上述結果本實驗方法應可適用於真實土壤樣品快速篩檢工具。

關鍵字

CALUX ； 土壤 ； Ad-DR bioassay ； 戴奧辛

並列摘要

Dioxins are recognized as endocrine disruptors in the environment. Dioxins have been raising public concern because of their highly toxic potential associated with several adverse health effects including skin lesions, impairment of the immune system, the delayed development of nervous system, interference with the endocrine system and reproductive functions, and cancers. A high-resolution gas chromatography coupled with a high-resolution mass spectrometry (HRGC/HRMS) is currently the most general method to determine dioxin concentrations, but HRGC/HRMS is not the most less-cost less and time-saving method compared to the other methods. Therefore, Chemically Activated LUciferase gene eXpression(CALUX) was widely used to analyze dioxin contamination such as environmental samples, food and provender so on. However, the HRGC/HRMS method requires expensive equipment and highly trained analysts, while the sample preparation procedures are often time consuming and expensive. Therefore, we need a fast and low cost analytical method for the large-scale dioxin surveillance. Thus, we used adenovirus infecting rat hepatoma cell (Ad-DRE-Luc/H4IIE) as a dioxin bioassay (Ad-DR bioassay). In this study we developed Ad-DRE-Luc/H4IIE cell lines which are such similar to CALUX system unlabeled standards of 2,3,7,8-TCDD, Certified Reference Materials(CRM) (Dioxins and Furans in Reference Sediment DX-1), and soil samples were tested. In order to enhance luciferase activation, we increased dioxin response elements from 4X to 6X and the culture incubation temperature was changed from 37 to 33°C during the induction period. By changes in our bioassay, the stability of bioassay had been improved. The TCDD-induced luciferase activation was increased in soil samples compared with the unchanged control. The detection limit of Ad-DR bioassay is 3.2 pM, and the correlation between HRGC/HRMS and Ad-DRE-Luc/H4IIEin the samples of soil was R2=0.6614. Finally, the results of this study were shown that the lowering incubation temperature had an effect on the enhancement of TCDD-induced luciferase activation in Ad-DRE-Luc/H4IIE cells. Ad-DR bioassay might be a useful tool for rapid screen of soil samples.

並列關鍵字

CALUX ； soil ； Ad-DR bioassay ； dioxin

參考文獻

郭彥祐，2011，利用新穎生物快速篩檢法檢測台灣土壤與底泥樣本中戴奧辛濃度，碩士論文，國立屏東科技大學，環境工程與科學系研究所，屏東。

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