亞硝酸是自然水生環境中即存在的陰離子，當環境中硝化與反硝化作用受影響而失衡時，亞硝酸則較為容易累積。在養殖方面，集約化的養殖方式將會快速的產生含氮廢物，如此便將提高了累積亞硝酸的風險。由過去的研究已知亞硝酸會對魚類造成許多生理功能的干擾，所以藉由分子層次的反應，來探討魚類面臨非致死且無行為異常之亞硝酸濃度時的逆境生理反應，則是相當重要的課題。當細胞遇到環境逆境時，將藉由活化熱休克反應(heat shock response; HSR)來進行細胞防衛的機制，本實驗利用吳郭魚做為研究物種，探討四個主要參與調節熱休克反應之基因：heat shock factor 1 (HSF1)、inducible heat shock protein 70 (HSP70)、heat shock cognate 70-1 (HSC70-1)、HSC70-2，在鰓、肝臟與肌肉中的mRNA表現量，以及在面臨亞硝酸曝露時的調節反應。實驗結果顯示四個基因在鰓上皆有最高的表現量，推測是由於魚類的鰓是直接與外界環境接觸的且具有多重生理功能的器官，因此其細胞防衛機制的調節較為活化。另一方面，在面臨亞硝酸逆境刺激時，僅發現在肌肉其HSP70的表現量下降之外，熱休克反應調節基因在三個研究之器官皆有顯著性上升的趨勢。進一步也發現HSC70-2於三個器官的變化幅度最大，為吳郭魚面臨亞硝酸逆境時反應最靈敏之基因。綜合以上實驗結果證實熱休克反應在魚類面臨亞硝酸刺激時是重要的抗逆境反應機轉，且具有潛力可運用於環境逆境的評估上。

Nitrite is a naturally existing anion in aquatic environments and imbalance of nitrification and denitrification occurs, leading to nitrite accumulation. Moreover, intensive recirculating aquaculture systems lead to high risk of elevation of nitrite concentration that causes the stressful effects on aquaculture species. However, studies on stress physiological responses of aquatic animals at molecular level upon nitrite stress are limited. In this study, an aquaculture species, tilapia (Oreochromis mossambicus), were used to examine mRNA expression of heat shock factor 1 (HSF1), inducible heat shock protein 70 (HSP70), heat shock cognate 70-1 (HSC70-1) and HSC70-2 in gills, liver and muscle to explore the regulatory response of cellular heat shock response (HSR), a crucial cytoprotective mechanism, to nitrite stress in fish. Our data showed that mRNA abundance of all studied genes was highest in gills. It reflected that the cytoprotection is highly demanding in fish gills since they directly contact external environment and possess multi-physiological functions. Furthermore, nitrite exposure significantly induced the transcript levels of HSF1 and all HSP70 isoforms in studied tissues except muscle HSP70. Importantly, our finding revealed that HSC70-2 is the most sensitive gene to nitrite exposure in various organs among the studied genes. To our knowledge, this is the first study to investigate HSF1 and three HSP70 isoforms simultaneously in fish exposed to nitrite stress. The present study provided the insight of cytoprotective role of HSR into nitrite stress as well as the potential ways to evaluate the risk of chronic nitrite exposure in fish.

目 錄
論文審定書 i
謝辭 ii
中文摘要 iii
英文摘要 iv
壹、前言 1
貳、材料和方法 5
2.1 魚種及飼養環境 5
2.2 亞硝酸暴露實驗 5
2.3 實驗目標的收集 5
2.4 總核醣核酸萃取和反轉錄實驗(total RNA extraction and Reverse Transcription) 6
2.5 即時定量聚合酶連鎖反應(Real-time PCR) 6
2.6 統計方法 7
參、結果 8
3.1 熱休克反應調節基因於實驗的目標組織的表現量 8
3.2 吳郭魚面臨亞硝酸暴露時其鰓上熱休克反應調節基因的表現量變化 8
3.3 亞硝酸暴露對於吳郭魚肝臟的熱休克反應調節基因表現量的影響 8
3.4 亞硝酸暴露對於吳郭魚肌肉的熱休克反應調節基因表現量的影響 9
肆、討論 10
參考文獻 15
附圖 23
附表 27
附錄 28

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