- 學位論文
利用斑馬魚不同基因之表現量對2,3,7,8-TCDD之檢測與比較
Comparison of the zebrafish's gene expression by 2,3,7,8-TCDD
摘要
隨著工業的發展，當今人們的周遭充斥著各式各樣的戴奧辛汙染，對於戴奧辛的全面普查是現今非常重要的課題，由於傳統的化學分析法耗費的時間長而且分析費用昂貴，趕不上緊急事件所要求的時效性，因此快速的生物檢測法逐漸受到大家的矚目，雖然生物檢測法無法提供準確的戴奧辛實際濃度，但在重大事件發生時，生物檢測法可以在短時間內判定事件發生範圍及程度，提供後續精準的化學分析測試一個有效性的前測參考，將可大大降低事件所帶來的衝擊。 CYP1A基因在戴奧辛檢測上已經被廣泛的探討，但斑馬魚P450家族及原致癌基因利用RT-PCR量化進行戴奧辛檢測的資料相對較少，所以本實驗選擇了斑馬魚的八種基因CYP1A、CYP1B、CYP2V1、CYP2AD3、CYP3C1、CYP3A65、tp53、MDM2，經過戴奧辛標準品處理過後，利用RT-PCR檢測八種基因的表現量，從八種基因中選取表現量較高的基因進行檢量線測試，其中只有CYP1A、CYP1B以及CYP3A65可以製作成檢量線，其R2值分別為0.992、0.991、0.968，再利用檢量線檢測環境中的戴奧辛樣品去做這三種基因的準確度比較。 結果顯示CYP1B最接近化學分析的結果，與化學分析比較有較為相近之倍數介於2.02~12.7，CYP1A與化學分析比較之倍數介於3.45~14.8，CYP3A65與化學分析比較之倍數介於0.012~0.059 由於每個基因對戴奧辛的敏感度不同，部分基因對戴奧辛的偵測反應不佳，能檢測的濃度範圍受限，若要利用斑馬魚基因進行戴奧辛檢測，目前還是以CYP1A表現量最好敏感度也最佳，其他基因尚待探討。
並列摘要
With the development of industry, people are surrounded by various kinds of pollutants, which causes adverse health effect. Dioxin is one of the most concerned pollutants. Therefore, the detection and analysis of dioxin is the major concern of researches.Traditional measurement of dioxin is time-consuming and costly which can’t fit the tight schedule for emergent case.Thus, a rapid bioassay on dioxin detection has been recently developed, which will provide fast analysis in short time.Although biological dioxin analysis unable to provide accurate dioxin concentration, it can be as a prescreening tool. CYP1A genes have been extensively studied in dioxin analysis recently, but not much work has been done on zebrafish P450 family and proto-oncogenes carried by RT-PCR quantification of dioxin detection. In this study, eight genes in zebra fish were selected for dioxin analysis: CYP1A, CYP1B, CYP2V1, CYP2AD3, CYP3C1, CYP3A65, tp53, MDM2. After dioxin standard treatment, RT-PCR was used for the detection of eight gene expressions. Higher expression levels of selected genes will be chosed to process following calibration curve tests.The results showed that only CYP1A, CYP1B and CYP3A65 can fit calibration curves with R2 values of 0.992, 0.991 and 0.968. To compare the results between chemical analysis and bioassay, the ratio of the result of CYP1A to chemical analysis ranged from 2.02 to 12.7，3.45 to 14.8 of CYP1B and 0.012 to 0.059 of CYP3A65.Zebra fish genetic test on dioxin analysis confirmed that CYP1A exhibited the best gene expression with high sensitivity. Other genes warrant futher investigation in the future.