利用定量即時聚合酶連鎖反應偵測斑馬魚CYP1A基因表現量之研究

Application of Q-PCR on zebrafish CYP1A gene expression

陳建宏(Jian-Hong Chen)

指導教授 ： 王雅玢

中原大學/工學院/土木工程研究所/碩士(2013年)

https://doi.org/10.6840/cycu201301067

摘要

戴奧辛對於脂肪有相當高的親和力，受汙染的環境透過食物鏈，累積在各式各樣的生物脂肪組織中，藉由生物累積進而影響人體。戴奧辛是一種非常穩定的化合物，如果遭受汙染，需要的復原時間很長且非常昂貴；分析戴奧辛使用一般傳統化學分析方法花費時間長，並且費用昂貴，生物檢測法雖然無法精準測量戴奧辛，但可以作為樣品初步篩選工具，省去過濾大量樣品之時間。
斑馬魚在研究上是一種良好的脊椎動物模型，因為斑馬魚的重複性與再現性非常好且生長快速，因此在許多研究中已經利用斑馬魚來作為研究模型。本研究利用斑馬魚CYP1A基因對於戴奧辛的表現量和定量Q-PCR對斑馬魚CYP1A基因之相對表現量，來初步估算樣品戴奧辛濃度，希望藉此達到初步篩選戴奧辛樣品之目的。
結果顯示在超音波直接濃縮萃取方法中，正己烷是表現相對較好的萃取溶劑，與化學分析比較有較為相近之倍數介於0.66~5.26倍之間，甲苯為2.35~26.8倍、正己烷/二氯甲烷為4.73~18.1倍，且經過管柱淨化之後縮小了與化學分析之誤差倍數，正己烷為0.42~4.56倍、甲苯為6.44~23.1倍、正己烷/二氯甲烷為6.75~17.9倍。在索氏萃取進行直接濃縮之萃取溶劑方面，甲苯是表現相對較好之萃取溶劑，與化學分析比較有較為相近之倍數介於4.45~8.23倍之間，正己烷為11.0~27.4倍、正己烷/二氯甲烷為1.9~35.3倍，經過淨化之後甲苯為10.06~60.45倍、正己烷為3.2~8.75倍、正己烷/二氯甲烷為1.44~22.9倍。
綜合整體結果，經過管柱淨化之樣品其生物分析與化學分析之趨勢較為相近，因此管柱淨化是必須存在的一個重要步驟。以斑馬魚Q-PCR分析方法來說，以正己烷當作萃取溶劑並利用索氏萃取法是最好的組合。

關鍵字

戴奧辛 ； 斑馬魚 ； 定量Q-PCR ； 飛灰 ； CYP1A ； 索式萃取 ； 超音波萃取

並列摘要

Polychlorinated dibenzo-p-dioxins and furans (PCDDs/Fs) or simply dioxins, are a group of organic polyhalogenated compounds that have been considered as environmental endocrine disruptors. They are bioaccumulated in humans and wildlife because of their lipophilic properties, and may cause developmental disturbances and cancer. Although the multicolumn cleanup procedure followed by high-resolution gas chromatography /high-resolution mass spectrometry (HRGC/HRMS) is widely used in PCDD/Fs analysis, this HRGC/HRMS method has several disadvantages including high consumption of time, energy, and cost. Therefore, a fast-screening test for dioxins is expected.
Zebrafish are an ideal vertebrate model system in which to study actions of TCDD on development because zebrafish embryos can be staged reproducibly and accurately. In this study, the quantification of Q-PCR on zebra fish was applied to study the CYP1A gene expression levels for dioxin and to be evaluated the possibility of being a fast-screening tool to dioxins analysis.
The results showed that n-Hexane is the better solvent in ultrasonic extraction without clean-up method. The ratio of bioassay to chemical analysis for n-Hexane, toluene and n-Hexane/dichloromethane ranged from 0.66 to 5.26, 2.35 to 26.8 and 4.73 to 18.1, respectively. The ratio of bioassay to chemical analysis after clean-up for n-Hexane, toluene and n-Hexane/dichloromethane ranged from 0.42 to 4.56, 6.44 to 23.1 and 6.75 to 17.9, respectively. Additionally, toluene is the better solvent in soxhlet extraction without clean-up method. In Soxhlet extraction and direct enrichment method, toluene is the better solvent. The ratio of bioassay to chemical analysis for n-Hexane, toluene and n-Hexane/dichloromethane ranged from 11.0 to 27.4, 4.45 to 8.23 and 1.9 to 35.3, respectively. The ratio of bioassay to chemical analysis after clean-up for n-Hexane, toluene and n-Hexane/dichloromethane ranged from 3.2 to 8.75, 10.06 to 60.45 and 1.44 to 22.9.
According to the result, the clean up process is necessary for dioxins analysis. For the zebrafish Q-PCR method, the result showed that n-Hexane as solvent with soxhlet extraction is the best combination.

並列關鍵字

Soxhlet extraction ； ultrasonic extraction ； Dioxin ； Zebrafish ； CYP1A ； Q-PCR ； Fly ash

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