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研究生: 彭慧文
Peng, Hui-Wen
論文名稱: 廣鹽性魚類適應鹽度變異中腸道與肝臟麩胺酸的運輸和代謝機轉
Glutamate transport and metabolism in intestine and liver of euryhaline teleost under ambient salinity perturbation
指導教授: 曾庸哲
Tseng, Yung-Che
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: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 76
中文關鍵詞: 廣鹽性魚類鹽度腸道肝臟麩胺酸尿素
英文關鍵詞: euryhaline teleosts, salinity, intestine, liver, glutamate, urea
DOI URL: https://doi.org/10.6345/NTNU202204128
論文種類: 學術論文
相關次數: 點閱:118下載:0
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  • 環境鹽度的變異已知會刺激廣鹽性魚類腸道中鈉鉀幫浦進行主動運輸以調節離子及水分的恆定;而肝臟則會進行代謝作用,以提供魚體各組織充足的能量需求。非必需胺基酸中的麩胺酸(glutamate)是生物體中含量最多的胺基酸,過去在魚類代謝機制相關的研究中,麩胺酸的運用已知與適應環境鹽度變異有關,然而麩胺酸在廣鹽性魚類腸道及肝臟的代謝調節機轉仍不明確。
      本研究運用日本種青鱂魚(Oryzias latipes)作為廣鹽性魚類實驗物種。在鹽度轉移過程中,腸道和肝臟中麩醯胺酸運輸蛋白(SAT1)及麩胺酸和麩醯胺酸相關的合成酵素(GLS, GLUL)基因表現亦隨著環境鹽度增加而有顯著變化,且上述隨鹽度變化波動的運輸蛋白和酵素皆被發現表現在腸道和肝臟細胞中。此外,鹽度刺激會使麩胺酸代謝產生毒性氨,因此本研究發現與尿素合成相關的基因在腸道(CPS, OTC)和肝臟(ASL, ARG, AGMAT)中會被誘發以進行尿素生合成反應。綜合以上的結果推測:鹽度刺激會促進廣鹽性魚類腸道和肝臟中麩胺酸與麩醯胺酸的代謝和運輸,因代謝產出的含氮廢物會藉由尿素循環機制合成尿素。此適應性代謝轉換機制不僅有利於個體能量的適時供給,並能有助於維持組織細胞內的滲透壓恆定。

    Environmental salinity variations have been proved to stimulate Na+-K+-ATPase (NKA) activity to regulate homeostasis in intestine of euryhaline teleosts. In addition liver can conduct metabolism for supplying intact energy demand. The non-essential amino acid, glutamate, is the most abundant amino acid in organisms. In euryhaline teleosts, glutamate accumulation and related metabolism have been observed in several vital tissues under salinity challenges. However, molecular evidences and related proper inferences regarding glutamate utilization are still in absence in teleost intestine and liver so far.
      In this study, we used Japanese medaka (Oryzias latipes) as a model euryhaline teleost for salinity perturbation test. Under salinity challenges, transcripts expressions of glutamine transporters (SAT1) and glutamate (Glu)/glutamine (Gln) synthesis enzymes (GSL and GLUL) in intestine and liver were obviously stimulated and found to be respectively expressed in intestinal epithelial cells and hepatocytes. Moreover glutamate deamination would further lead to NH4+ production; therefore, those NH4+-derived urea cycle candidates, carbamoyl phosphate (CPS) and ornithine transcarbamylase (OTC) in intestine, argininosuccinate lyase (ASL), arginase (ARG) and agmatinase (AGMAT) in liver, were found to be up-regulated under ambient salinity challenges. Those results inferred that Glu/Gln metabolism, subsequent transport and further urea cycle activation from metabolic nitrogenous waste in intestine and liver of euryhaline teleosts may not only be vital for intact metabolic demand, but also for body osmolality maintenance in face of salinity challenges.

    致謝 i 摘要 ii Abstract iii 目錄 v 研究背景 1 廣鹽性魚類的鹽度適應機制 1 廣鹽性魚類在鹽度適應過程的能量代謝機轉 3 (1) 鹽度適應過程個體的耗氧與基礎代謝 3 (2) 鹽度適應過程的能量供應途徑 4 (3) 鹽度適應過程的麩胺酸相關代謝機制 6 動物細胞的麩胺酸-麩醯胺酸運輸調節 8 鹽度適應過程中短期與長期的定義 9 研究目的 11 材料與方法 12 實驗動物飼養 12 (1) 實驗動物來源及馴養環境 12 (2) 實驗動物的鹽度處理 12 Total RNA 萃取 13 反轉錄聚合酶連鎖反應 (Reverse transcription reaction, RT) 13 即時定量反轉錄聚合酶連鎖反應 (Real-time quantitative polymerase chain reaction, RT-PCR) 14 石蠟包埋 (Paraffin-embedded sections) 14 (1) 石蠟包埋 14 (2) 石蠟切片 15 (3) 脫蠟 15 RNA 探針合成 (RNA probe synthesis) 16 原位雜合技術 (in situ hybridization) 16 免疫螢光染色 (Immunocytochemistry, ICC) 17 胺基酸含量分析 18 尿素含量分析 19 Glutamate dehydrogenase (GDH)活性分析 19 統計分析 20 結果一 21 鹽度刺激對青鱂魚腸道內麩胺酸代謝相關之胺基酸含量的影響 21 鹽度刺激對青鱂魚腸道內麩胺酸和麩醯胺酸之運輸蛋白的影響 21 鹽度刺激對青鱂魚腸道的麩胺酸和麩醯胺酸之合成酶的影響 23 青鱂魚腸道細胞內麩胺酸和麩醯胺酸相關基因位置 24 鹽度刺激對青鱂魚腸道Glutamate dehydrogenase (GDH)活性之影響 24 鹽度刺激對青鱂魚腸道中含氮廢物調節之影響 24 結果二 27 鹽度刺激對青鱂魚肝臟內麩胺酸代謝相關之胺基酸含量的影響 27 鹽度刺激對青鱂魚肝臟內麩胺酸和麩醯胺酸之運輸蛋白的影響 27 鹽度刺激對青鱂魚肝臟的麩胺酸和麩醯胺酸之合成酶的影響 28 青鱂魚肝臟細胞內麩胺酸和麩醯胺酸相關基因位置 29 鹽度刺激對青鱂魚肝臟Glutamate dehydrogenase (GDH)活性之影響 30 鹽度刺激對青鱂魚肝臟中含氮廢物調節之影響 30 討論 33 胺基酸對於廣鹽性魚類在鹽度適應過程的重要性 33 麩胺酸-麩醯胺酸對於廣鹽性魚類在鹽度適應過程中的重要性 35 鹽度適應對於廣鹽性魚類腸道和肝臟中麩胺酸-麩醯胺酸相關運輸蛋白和酵素的影響 36 (1) 腸道 36 (2) 肝臟 37 廣鹽性魚類在短期與長期的鹽度適應過程中,腸道和肝臟中麩胺酸/麩醯胺酸相關代謝機轉的差異 38 廣鹽性魚類在鹽度適應過程中腸道和肝臟含氮廢物的代謝 39 結論 42 參考文獻 43 附表 49 表一、RT-PCR/qRT-PCR引子序列設計 49 表二、In situ hybridization探針序列設計 51 附圖 52 圖一、轉移至FW、10‰ SW及20‰ SW後的6、24、72、168小時之青鱂魚(Oryzias latipes)腸道中胺基酸含量 52 圖二、鹽度刺激腸道麩胺酸運輸蛋白之相對基因表現量 53 圖三、鹽度刺激腸道麩醯胺酸運輸蛋白之相對基因表現量 54 圖四、鹽度刺激腸道麩胺酸合成酶之相對基因表現量 55 圖五、鹽度刺激腸道麩醯胺酸合成酶之相對基因表現量 56 圖六、青鱂魚腸道細胞內麩胺酸和麩醯胺酸相關基因位置 57 圖七、鹽度刺激對青鱂魚腸道中Glutamate dehydrogenase (GDH)活性之影響 58 圖八、鹽度刺激腸道氨運輸蛋白之相對基因表現量 59 圖九、鹽度刺激對青鱂魚腸道中氨含量的影響 60 圖十、鹽度刺激腸道尿素循環合成酵素之相對基因表現量 61 圖十一、鹽度刺激腸道尿素循環合成酵素之相對基因表現量 62 圖十二、鹽度刺激對青鱂魚腸道中尿素含量的影響 63 圖十三、轉移至FW、10‰ SW及20‰ SW後的6、24、72、168小時之青鱂魚(Oryzias latipes)肝臟中胺基酸含量 64 圖十四、鹽度刺激肝臟麩胺酸運輸蛋白之相對基因表現量 65 圖十五、鹽度刺激肝臟麩醯胺酸運輸蛋白之相對基因表現量 66 圖十六、鹽度刺激肝臟麩胺酸合成酶之相對基因表現量 67 圖十七、鹽度刺激肝臟麩醯胺酸合成酶之相對基因表現量 68 圖十八、青鱂魚肝臟細胞內麩胺酸和麩醯胺酸相關基因位置 69 圖十九、鹽度刺激對青鱂魚肝臟的Glutamate dehydrogenase (GDH)活性之影響 70 圖二十、鹽度刺激肝臟氨運輸蛋白之相對基因表現量 71 圖二十一、鹽度刺激對青鱂魚肝臟中氨含量的影響 72 圖二十二、鹽度刺激肝臟尿素循環合成酵素相對基因表現量 73 圖二十三、鹽度刺激肝臟尿素循環合成酵素相對基因表現量 74 圖二十四、鹽度刺激對青鱂魚肝臟中尿素含量的影響 75 圖二十五、廣鹽性硬骨魚類腸道與肝臟中麩胺酸和麩醯胺酸示意圖 76

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