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研究生: 孔耕心
Kung, Geng-Xin
論文名稱: 銀離子於酸性環境中對斑馬魚胚胎發育及離子調控之危害
Silver exposure impairs the development and ion regulation of zebrafish embryos in an acidic environment
指導教授: 林豊益
Lin, Li-Yih
口試委員: 洪君琳
Horng, Jiun-Lin
周銘翊
Chou, Ming-Yi
口試日期: 2021/07/27
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: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 66
中文關鍵詞: 銀離子酸環境斑馬魚離子細胞氧化壓力
英文關鍵詞: Silver ions, Acidic freshwater, Zebrafish, Ionocyte, Oxidative stress
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202101032
論文種類: 學術論文
相關次數: 點閱:44下載:2
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  • 奈米銀在近年來被廣泛地運用於醫療及化妝品產業,其廢棄材料表面釋放出的銀離子會危害水生生物。然而,目前對於銀離子在不同酸鹼值環境中的毒性差異並不瞭解。本研究利用斑馬魚胚胎為水生生物模式,將其暴露於含有0、0.1及0.25 ppm AgNO3之pH 5或pH 7環境中,以評估銀離子毒性的差異。在暴露96小時的結果中可以得知銀離子的胚胎發育影響程度隨著濃度升高而增加。在銀離子的暴露後胚胎體長顯著下降,而卵黃囊面積則會增加。銀離子亦導致胚胎耳石囊面積及耳石面積下降,影響胚胎的內耳系統。另外,側線神經丘及毛細胞數目下降也反映出銀離子對胚胎側線的影響。值得注意的是:酸環境(pH 5)中會增加銀離子在體長、卵黃囊、耳石囊以及耳石上的影響。另一方面,銀離子降低離子細胞開口面積及數目,阻礙胚胎的離子調節功能,並且導致NaRC及HRC數目顯著降低,增加胚胎適應酸環境的困難性。另外,30分鐘及2小時的實驗中可發現:銀離子會藉由增加離子細胞的氧化壓力程度,進而誘發細胞凋亡機制。總結以上結果,本研究認為酸環境會增加銀離子對魚類的危害。

    Silver nanoparticles are widely used in medical treatments and cosmetic industries. Ag+ released from the particles causes physiological damages to aquatic organisms. However, it is still unclear how environmental pH influences the toxicity of Ag+. In this study, zebrafish embryos were incubated in pH 5 or pH 7 water containing different concentrations of AgNO3 (0, 0.1 and, 0.25ppm) to examine the toxic effect of Ag+. After 96 h exposure, Ag+ influenced embryonic development in a dose-dependent manner. The body length, otic vesicle size, and saccular otolith size were significantly decreased, yet the yolk sac size was increased. Moreover, the neuromast and L1 hair cell numbers were reduced by Ag+. Acidic water (pH 5) enhanced the toxic effects of Ag+ on body length, size of yolk sac, otic vesicle, and saccular otolith. Ag+ also impaired ion regulation of embryos by reducing the apical opening size and cell number of ionocytes. The cell numbers of both NaRC and HRC were significantly decreased by Ag+. After shorter exposures (30 min and 2 h), Ag+ increased the oxidative stress of ionocytes and induced cell apoptosis. In conclusion, this study suggests that acidified environments might intensify the Ag+ toxicity to fish.

    摘要 1 Abstract 2 研究背景 3 奈米銀汙染 3 銀離子對魚類的危害 4 水環境酸化現象 5 魚類的酸適應機制 6 斑馬魚模式生物 6 斑馬魚毛細胞 7 斑馬魚離子細胞 7 氧化壓力與細胞死亡機制 8 研究目的 10 實驗設計流程圖 11 實驗設計 12 實驗一:96小時處理對斑馬魚胚胎死亡及發育之影響 12 1-1:觀察7天處理對各組斑馬魚胚胎死亡率之影響 12 1-2:觀察96小時處理對各組斑馬魚胚胎發育指標影響之差異 12 1-3:觀察96小時處理對各組斑馬魚胚胎內耳系統影響之差異 12 1-4:觀察96小時處理對各組斑馬魚胚胎側線系統影響之差異 12 實驗二:96小時處理對斑馬魚胚胎離子調控之影響 13 2-1:觀察96小時處理對各組斑馬魚胚胎離子細胞變化之影響 13 2-2:觀察96小時處理後各組斑馬魚胚胎離子調節差異 13 實驗三:探討銀離子損害斑馬魚胚胎離子細胞之機制 13 3-1:觀察30分鐘處理後斑馬魚胚胎離子細胞數目、氧化壓力程度及細胞凋亡訊號變化14 3-2:觀察2小時處理後斑馬魚胚胎離子細胞數目、氧化壓力程度及細胞凋亡訊號變化 14 材料與方法 15 一、實驗動物 15 二、馴養水體 15 三、斑馬魚胚胎死亡率計算 16 四、斑馬魚胚胎發育指標分析 16 五、斑馬魚胚胎活體螢光染色 16 六、免疫細胞組織染色 (Whole mount immunocytochemistry, ICC) 17 七、電感耦合電漿體質譜法 (Inductively coupled plasma mass spectrometry, ICP-MS) 18 八、掃描式電子顯微鏡 (Scanning electron microscopy, SEM) 18 九、實驗數據分析 18 結果 20 結果一:96小時處理對斑馬魚胚胎死亡率及發育之影響 20 1-1:斑馬魚胚胎累積死亡率 20 1-2:斑馬魚胚胎體長及卵黃囊面積 20 1-3:斑馬魚胚胎耳石囊及耳石面積 21 1-4:斑馬魚胚胎側線神經丘及L1毛細胞數目 22 結果二:96小時處理對斑馬魚胚胎離子調控之影響 23 2-1:斑馬魚胚胎離子細胞頂膜開口面積 23 2-2:斑馬魚胚胎離子細胞型態及數目 23 2-3:斑馬魚胚胎NaRC細胞型態及數目 24 2-4:斑馬魚胚胎HRC細胞數目 25 2-5:斑馬魚胚胎體內銀、鈉及鈣離子含量 25 結果三:30分鐘及2小時處理胚胎離子細胞之變化 26 3-1:30分鐘處理對斑馬魚胚胎離子細胞之變化 26 3-2:2小時處理對斑馬魚胚胎離子細胞之變化 27 討論 29 銀離子對斑馬魚胚胎死亡率之影響 29 銀離子對斑馬魚胚胎發育之影響 29 銀離子對斑馬魚胚胎聽覺系統及側線系統之影響 30 銀離子降低斑馬魚胚胎的酸適應功能 30 不同酸鹼值環境中銀離子累積量的差異 31 銀離子對NaRC及HRC影響之差異 32 銀離子阻礙斑馬魚胚胎的鈉及鈣離子吸收功能 33 銀離子增加離子細胞氧化壓力誘導細胞凋亡 33 環境的意義 34 結論 36 參考文獻 37

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