EPA-100-1604-02-01

100

1,960 千元

2011/07/25

2011/12/31

趙浩然

環檢所

陳元武

國立屏東科技大學(環境工程與科學系)

環境監測及檢測

類戴奧辛化合物、化學活化冷光酵素基因表現法

Dioxin-like compounds, CALUX

鄒粹軍;王雅玢

趙浩然

08-7703202#7517

chao.hr@gmail.com

目前國內環境與生物檢體的類戴奧辛化合物濃度檢測，大多利用高解析氣相層析質譜儀(HRGC/HRMS)進行檢測，該檢測技術已相當成熟且準確，但過程耗費時間且分析成本偏高，以致無法應用HRGC/HRMS對環境背景值進行大樣本數的調查。近年來在歐、美、日等已開發國家，均利用化學活化冷光酵素基因表現法(CALUX)為戴奧辛快速篩檢方法，以降低社會所付出的戴奧辛檢驗成本。延續98、99年度環檢所計畫，以人類肝腫瘤Huh7-DRE-Luc與大鼠肝腫瘤Ad-DRE-Luc/H4IIE細胞株之研究成果為基礎，本團隊嘗試將Huh7-DRE-Luc的第一代細胞株降低暴露戴奧辛溫度到35℃，並在冷光施測前6-8小時添加PMA 8μM至medium中，稱為new Huh7-DRE-Luc system，可得到冷光數值顯著上升，並將工作範圍由100 pM(第一代)降至50 pM(new Huh7-DRE-Luc system)，且50 pM之後RLU值皆比未降溫及加藥時高出5~10倍。同時，將第二代大鼠肝腫瘤Ad-DRE-Luc/H4IIE細胞株由4倍DRE提升為6倍DRE(第三代細胞株)，並配合暴露戴奧辛溫度至33℃但不添加任何藥物，本設計可增強細胞株的穩定度至RSD<15%，工作範圍為10~30 pM之間。此兩項結果在搭配希爾方程式(Hill curve)配適2,3,7,8-TCDD檢量線後，也有合乎法規的配適度。本計畫的研究成果如下：(1)引進環檢所開發出之快速前處理標準方法程序（包含索氏萃取、CAPE酸性矽膠-活性碳複合管柱），已建構完成；(2)完成細胞分析方法標準QA/QC程序（期末報告4-1節）；(3)以Hill curve公式配適2,3,7,8-TCDD檢量線的R2均可達到0.98以上，符合法規標準；(4)土壤底泥樣品已由13個樣本中(土壤:6個；底7個)完成70件次(99年度樣品)，其中土壤樣品第一代細胞株與new Huh7-DRE-Luc system的DRE-luciferase/GC-HRMS比值可由25.7倍降至7.28倍(GC-HRMS測值< 100 ng I-TEQ/kg)；11.6倍下降至3.46倍(GC-HRMS測值> 100 ng I-TEQ/kg)。底泥樣品的第一代細胞株與New Huh7-DRE-Luc system的DRE-luciferase/GC-HRMS比值可由26.0倍(8X)降至4.09(1X)倍(樣本的GC-HRMS測值> 100 ng I-TEQ/kg d.w.)；(5)本計畫第二代細胞(Ad-DRE-Luc/H4IIE)的效能與上年度計畫的成效相當；(6)new Huh7-DRE-Luc檢測3個生物體樣本25件次，平均比值為9.58倍，Ad-DRE(6X)-Luc/H4IIE檢測5個生物體樣本22件次，Ad(6X)/GC-HRMS比值可比第二代細胞株由9.33倍降至5.89倍(GC-HRMS測值< 1 ng I-TEQ/kg w.w.)；7.79倍下降至1.67倍(GC-HRMS測值> 1 ng I-TEQ/kg w.w.)。最後，感謝環檢所提供本團隊三年之研究經費。希望未來可利用new Huh7-DRE-Luc system與Ad-DRE(6X)-Luc/H4IIE 並搭配適合的硫酸矽膠濃度配比，對土壤、底泥、魚體等環境檢體進行快速的類戴奧辛化合物的篩檢結果，節省國家社會的開支。

類戴奧辛化合物、化學活化冷光酵素基因表現法

污染場址國內新戴奧辛生物快速篩選檢測技術開發計畫(II)期末報告.pdf

11MB

污染場址國內新戴奧辛生物快速篩選檢測技術開發計畫(II)期末報告

2012/2/1 上午 12:00:00

Previously, we have established two recombinant hepatoma systems including a recombinant hepatoma cell (Huh7-DRE-Luc ,the first generation cell) a recombinant adenovirus carrying a DRE-driven luciferase gene and using a rat hepatoma celll line H4IIE as the host (Ad-DRE-Luc/H4IIE,the second generation cell). On the basis of this DRE-driven luciferase assay system , this project aims to develop a high-through put screening bioassay for dioxin-like compounds measurements in various matrices (e.q. soils, sediments, and bio-sample). In the result of the present study, a fast cleanup system and the standard cell culture procedure for dioxin-bioassay have been set up. After fulfilled to QA/QC criteria and filled into the calibration curve (i.e. Hill equation), the significant regression model of 2,3,7,8-TCDD was shown with R2≧0.995. We established a more high-throughput and high-sensitive recombinant system (Ad-DRE(6X)-Luc/H4IIE, the third generation)compared to the previous system (Huh7-DRE-Luc and Ad-DRE-Luc/H4IIE).The first generation system (Huh7-DRE-Luc cells) was tested by the change of incubation temperature (35℃) and the addition of PMA(Phorbol-12-myristate-13-acetate) to be a new system, namely new Huh7-DRE-Luc system. These new two systems were tested with a liquid standard (EDF-5416), a certified reference material DX-1, soils, sediments, and bio-samples. One-hundred-and-forty-six samples were examined in our new bioassay systems. In conclusion, the sensitivity of new Huh7-DRE-Luc and Ad-DRE(6X)-Luc/H4IIE systems in dioxins measurement are significant higher than those of the previous systems including the first and second generation cells. The new systems (new Huh7-DRE-Luc system and Ad-DRE(6X)-Luc/H4IIE have been demonstrated to be a powerful technique for pre-screening and fast-screening dioxin samples from various sources, including organisms.

Dioxin-like compounds, CALUX