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研究生: 趙仕堯
Chao, Shih-Yao
論文名稱: 缺氧誘導因子與廣鹽性硬骨魚類滲透壓生理調節之關聯
Linkage of the HIF-1 and osmoregulatory significance in euryhaline teleost
指導教授: 曾庸哲
Tseng, Yung-Che
林豊益
Lin, Li-Yih
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2017
畢業學年度: 105
語文別: 中文
論文頁數: 64
中文關鍵詞: 缺氧誘導因子鹽度轉移廣鹽性魚類鈉鉀幫浦
英文關鍵詞: Hypoxia-inducible factors, salinity challenges, euryhaline teleost, Na+/K+-ATPase
DOI URL: https://doi.org/10.6345/NTNU202202233
論文種類: 學術論文
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  • 穩定體液滲透度是廣鹽性魚類在遭遇環境鹽度變異時重要的生理適應策略。而眾多非生物性環境擾動均會影響生物缺氧誘導因子(HIFs)蛋白功能的穩定性,進而影響細胞運作。然而目前對於魚類面臨滲透壓波動挑戰時,細胞內HIFs相關訊息網絡與滲透壓恆定的研究仍非常缺乏。本研究選用India medaka (Oryzias melastigma)作為暴露高鹽環境的廣鹽性硬骨魚實驗物種,實驗結果發現其仔魚在面臨鹽度上升刺激時,HIF-1α的基因和蛋白質表現量皆會顯著上升,而部分HIF-1α的核酸訊號亦會表現在仔魚表皮的離子細胞。此外,HIF-1α蛋白的弱化處理,除了會顯著地影響仔魚鈉鉀幫浦的蛋白表現量,亦會降低其在高鹽處理時表皮對於鈉與氯離子的排放;然而,仔魚表皮上離子細胞的表現密度,並未受到HIF-1α蛋白的弱化影響。我們據此推論:HIF-1α於表皮細胞的表現,有助於廣鹽性魚類在面臨環境鹽度挑戰時進行體液調節機制。

    Maintenance of osmostasis is one of the most important physiological processes for euryhaline teleosts to cope with ambient salinity challenges. And hypoxia-inducible factors (HIFs) were found to be involved in several cellular regular functions under diverse abiotic perturbations. However, HIFs-related signaling for osmostasis in euryhaline teleosts under osmotic challenges are still unknown.
    In this study, India medaka (Oryzias melastigma) was utilized as an euryhaline teleost model to study the possible roles of HIFs for hyperosmotic adaptation. Based on eveluations of transcripts and protein expressions, ambient salinity challenges apparently up-regulated HIF-1 expressions in madaka larvae, and HIF-1 homologue (hif-1al) was partially expressed in Na+/K+-ATPase (NKA)-positive epithelial cells. Moreover, abrogation of HIF-1α-like obviously depressed NKA protein expression as well as sodium and chloride effluxes from yolk sac epithelium in seawater condition, while NKA-positive cell density on epithelium was not changed in HIF-1α-like morphants in comparison to mismatch control ones. Consequently, HIF-1α expression and stabilization in epithelium are essential for maintaining osmostasis in euryhaline teleosts under salinity challenges.

    摘要 1 Abstract 2 目錄 4 前言 8 鹽度對於魚類生長發育的影響 8 廣鹽性魚類對於鹽度的適應 9 環境壓力因子如何調節魚類面對環境緊迫 10 缺氧誘導因子(HIFs) 11 缺氧誘導因子對水生生物的影響 13 實驗目的 14 材料與方法 15 一、 實驗動物飼養 15 一之一、 實驗動物飼養 15 一之二、 實驗動物的鹽度處理 15 二、 青鱂魚胚胎基本發育的測量 16 三、 耗氧速率偵測 16 四、 Total RNA 萃取 17 五、 反轉錄聚合酶連鎖反應 (Reverse transcription reaction, RT) 17 六、 及時定量反轉錄聚合酶連鎖反應(Real-time quantitative polymerase chain reaction, RT-PCR) 18 七、 西方墨點法 (Western blotting) 19 七之一、 蛋白質樣本備制 19 七之二、 缺氧誘導因子 (Hypoxia-inducible factor-1 alpha, HIF-1α)蛋白檢測 19 七之三、 鈉鉀幫浦 (Na+/K+-ATPase, NKA)蛋白檢測 21 八、 原位雜合反應 (in situ hybridization) 21 八之一、 基因選與定序 21 八之二、 全胚胎原位雜合反應 (whole mount in situ hybridization) 22 九、 抑制基因轉錄 23 十、 掃描式離子選擇電極技術(scanning ion selective electrode technique, SIET) 23 十之一、 將青鱂魚胚胎浸泡hatching enzyme破殼 23 十之二、 使用SIET測量青鱂魚胚胎在鹽度轉移下之排鈉氯情形 24 十一、 細胞免疫螢光染色 (Immunocytostaining) 26 結果 27 一、 環境鹽度變異對青鱂魚胚胎發育之影響 27 二、 環境鹽度變異對青鱂魚胚胎耗氧代謝的影響 27 三、 青鱂魚胚胎面臨鹽度刺激時體內環境壓力因子的表現 28 四、 鹽度緊迫對青鱂魚胚胎體內缺氧誘導因子蛋白的影響 29 五、 青鱂魚胚胎體內缺氧誘導因子的空間表現相 29 六、 抑制hif-1al基因的轉錄機制 30 六之一、 胚胎死亡率統計 30 六之二、 青鱂魚胚胎在鹽度30‰海水中鈉與氯的排出檢測 31 六之三、青鱂魚胚胎在鹽度30‰海水中表皮離子細胞的表現 31 六之四、青鱂魚胚胎在在鹽度30‰海水中的基礎代謝率 32 討論 33 一、 鹽度對於海洋青鱂魚(Oryzias melastigma)胚胎發育以及基礎代謝率的影響 33 二、 缺氧誘導因子(Hypoxia-inducible factors)及相關壓力因子對於廣鹽性硬骨魚類面臨鹽度刺激是否表現及作用之探討 34 三、 缺氧誘導因子(Hypoxia-inducible factors)的表現與廣鹽性硬骨魚類滲透壓適應之關聯 36 結論 38 參考文獻 39 附表 48

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