| 摘要: | 本研究目的在於利用已屆淘汰年齡，但仍具備產蛋能力之白色來亨雞(White Leghorn)，先以戴奧辛受體(Aryl hydrocarbon receptor, AHR)基因之引子(Primer)，以反轉錄－聚合酶連鎖反應( RT-PCR)分析禽類體內之肝、脾、肺、腎、粒性細胞、膜性細胞(Theca)、輸卵管(Fallopian tube)、殼腺(Shell gland)、肌肉與血液等組織或器官之AHR基因表現情形，以選出對戴奧辛有較佳感受性之器官，並挑選出取得容易、可大量製備、處理簡易、經濟且易於培養之組織，進一步比較其細胞色素P450A(Cytochrome P450A,)基因表現，篩選出適當組織來建立一個經濟且快速之戴奧辛類物質生物檢測方法。結果顯示，受測動物的肝、腎、卵巢濾泡粒性細胞與血液中明顯表現AHR，而粒性細胞擁有取得容易、易於培養及保存之特性，所以將使用粒性細胞做為生物檢測系統之主要材料。粒性細胞之CYP1A4基因表現與戴奧辛刺激濃度成正向反應，但細胞存活率則與戴奧辛濃度成反比。Real-time PCR結果顯示，在戴奧辛刺激八小時後，粒性細胞的CYP1A4基因表現紊亂，其穩定性不高，只在戴奧辛濃度為10-3～101nM呈現劑量反應，而在戴奧辛濃度為10-3～102nM與CYP1A4基因表現量之決定係數達0.931，得知戴奧辛濃度在此範圍內，可被此系統所偵測並定量。
本研究目前之實驗結果可應用於戴奧辛類化合物之定性及半定量分析，並且於戴奧辛刺激細胞八小時內可應用於特定濃度範圍之戴奧辛(3ppt～30ppb)。定量分析若能進一步提高此系統之穩定度與偵測極限，則可做為替代現有之戴奧辛生物檢測系統之另一選擇。
This study was designed to utilize the retired laying hens for constructing a bioassay system for environmental hormones. First used reverse transcription-PCR (RT-PCR) to analysis aryl hydrocarbon receptor (AHR) gene expression in liver, spleen, lung, kidney, granulosa, theca, fallopian tube, shell gland, muscle and blood of the animal, then to select organs which has a better sensitivity for dioxin detection, easy to obtain and prepare, economical and easy to culture for the establishment of an economic and rapid detection method for the Dioxin-like compounds. The results showed that the liver, kidney, ovarian granulosa cells and blood exhibit AHR gene expression. Among all, granulosa cells are easy to culture and store, so this study will use the granulosa cells for the biological detection system. There is a positive correlation between dioxin stimulating doses and the CYP1A4 gene expression in granulosa cells. However, the cell survival rate is inversely related with the concentration of dioxin. Real-time PCR results show that the CYPA4 gene expression in granulosa is not stable after dioxin stimulation for 8 hours. Gene expression was obvious between 10-3 ~ 101nM of dioxin. There is a good linear response in the 10-1 ~ 102nM (correlation coefficient = 0.931). In conclusion, this system can be applied to the qualitative analysis of dioxin compounds, and eight hours of stimulating time is recommended. The detection range is between 3ppt ~ 30ppb. Further study is needed to improve the stability and detection limits of dioxin in order for this system to be an alternative bioassay system. |
