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研究生:李致穎
研究生(外文):Lee, Chih-Ying
論文名稱:以斑馬魚為動物模式研究奈米金屬對離子細胞之毒性
論文名稱(外文):Using Zebrafish as a Model Animal to Investigate the Toxicities of Nanometals to Ionocytes
指導教授:林豊益洪君琳
指導教授(外文):Lin, Li-YihHorng, Jiun-Lin
學位類別:博士
校院名稱:國立臺灣師範大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:70
中文關鍵詞:奈米銀顆粒奈米銅顆粒斑馬魚離子細胞毒性
外文關鍵詞:AgNPCuNPzebrafishionocytetoxicity
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在21世紀,奈米科技快速發展,應用廣泛,而這也同時增加了人類接觸各種不同奈米顆粒的機會,因此針對其毒性的研究,也相形越來越重要。而其中,金屬奈米顆粒對於魚類離子細胞的潛在毒性仍未有充分的研究。本篇研究是以斑馬魚胚胎為動物模式,探討奈米銀和奈米銅對斑馬魚胚胎皮膚上的離子細胞功能之毒性作用。實驗方式是將斑馬魚的胚胎分別浸泡於奈米銀和奈米銅96小時(受精後4〜100小時(hpf))後,檢測其全身離子含量及皮膚上離子細胞的數量和功能。暴露於奈米銀96小時後,全身Na離子和K離子含量在奈米銀濃度為3 mg/L組別中顯著下降,而Ca離子含量在濃度 ≥ 0.1 mg/L時下降。以Scanning ion-selective electrode technique(SIET)檢測胚胎皮膚的H離子分泌功能,發現在3 mg/L時功能顯著降低。以rhodamine 123(粒線體標記)來標記離子細胞,其密度在1和3 mg/L時分別降低了25%和55%,而離子細胞外觀也從橢圓形變形為棘狀形。進一步以抗體標記染色的方式檢測不同離子細胞亞型,發現奈米銀對富含H+-ATPase的HR細胞和富含Na+/K+-ATPase的NaR細胞造成不同的損傷。進一步使用掃描電子顯微鏡觀察離子細胞,其頂端開口有明顯的萎縮,這和正常功能的喪失相關。另一方面,斑馬魚胚胎暴露於奈米銅後,觀察到相同的趨勢。濃度在 ≥ 0.1 mg/L時,全身Na離子和Ca離子含量顯著降低,而在 ≥ 1 mg/L時,K離子含量降低。而濃度 ≥ 1 mg/L時,胚胎皮膚的H離子排泄功能顯著降低。在奈米銅濃度 ≥ 0.1 mg/L時,用rhodamine 123標記的活離子細胞數量顯著減少。使用掃描電子顯微鏡觀察離子細胞,其頂端開口同樣有明顯的萎縮。我們也以免疫染色方式進行離子細胞亞型(HR細胞和NaR細胞) 標記,兩者都在濃度 ≥ 1 mg/L時降低。透過檢測離子轉運蛋白/通道和鈣離子調節激素mRNA表現量,發現功能的損傷也藉由基因表達的變化反映出來。綜合以上結果證實在斑馬魚胚胎早期,奈米銀和奈米銅對其皮膚離子細胞會產生毒性和並影響其離子調節的功能。由於斑馬魚離子細胞和人類腎臟細胞,在生理功能上和對環境變化的調節反應上都有高度的相似,故此一結果也提醒我們奈米金屬對人類的腎臟細胞可能有潛在毒性。
Nanotechnology is a new research area that is explosively growing across a wide range of industries. It extensively appears in our daily lives in many fields. However, The potential toxicity of metals nanoparticles to the ionocytes of fish is still unclear. This study used zebrafish embryos as a model to investigate the toxic effects of AgNPs and CuNPs on ion regulation by skin ionocytes. Zebrafish embryos were exposed to AgNPs or CuNPs for 96 h (4~100 h post-fertilization (hpf)). After 96 h of exposure to AgNPs, whole-body Na+ and K+ contents significantly decreased at 3 mg/L, while Ca2+ contents decreased at ≥ 0.1 mg/L. H+ secretion by the skin significantly decreased at 3 mg/L. The density of skin ionocytes labeled with rhodamine 123 (a mitochondrion marker) decreased by 25% and 55% at 1 and 3 mg/L, respectively; and 54% of ionocytes (at 3 mg/L) were deformed from an oval to a spinous shape. We also used immunocytochemistry to label two major subtypes of ionocytes, the H+-ATPase-rich ionocytes (HR cells), and Na+/K+-ATPase-rich ionocytes (NaR cells). The number of HR cells significantly decreased by 30% and 41% in 1 and 3 mg/L AgNP groups, respectively; and the apical opening of HR cells became smaller. In contrast, the number of NaR cells significantly increased by 29% and 43% in 1 and 3 mg/L groups, respectively, while these cells deformed from an oval to a spinous shape. After exposure to CuNPs, whole-body Na+ and Ca2+ contents were significantly reduced at ≥ 0.1 mg/L, while the K+ content had decreased at ≥ 1 mg/L. H+ excretion by the skin significantly decreased at ≥ 1 mg/L. The number of living ionocytes labeled with rhodamine123 (a mitochondrion marker) had significantly decreased with ≥ 0.1 mg/L CuNPs. The ionocyte subtypes of H+-ATPase-rich (HR) and Na+/K+-ATPase-rich (NaR) cells were also labeled by immunostaining and had decreased with ≥ 1 mg/L. Shrinkage of the apical opening of ionocytes was revealed by scanning electronic microscopy. Functional impairment was also reflected by changes in gene expressions, including ion transporters/channels and Ca2+-regulatory hormones. This study revealed the toxicity of AgNPs and CuNPs to skin ionocytes and ion regulation in the early stages of zebrafish embryos. The damage to zebrafish ionocytes by AgNPs and CuNPs could also be a warning to human beings, because ionocytes are functionally similar to our renal epithelial cells.
1. 研究背景 1
1.1. 現今的奈米科技 1
1.2. 奈米銀顆粒的毒性 2
1.3. 奈米銅顆粒的毒性 4
1.4. 以斑馬魚為動物模式 5
1.5. 斑馬魚的離子細胞 6
1.6. 奈米顆粒的毒性機轉 8
2. 研究目標 10
2.1. 探討奈米銀顆粒對斑馬魚胚胎時期的離子細胞造成的毒性傷害 10
2.2. 探討奈米銅顆粒對斑馬魚胚胎時期的離子細胞造成的毒性傷害 10
3. 研究材料與方法 12
3.1. 斑馬魚 12
3.2. 奈米銀顆粒的暴露 12
3.3. 奈米銅顆粒的暴露 13
3.4. 整體胚胎離子含量測量 13
3.5. 離子細胞染色及數量測定 14
3.6. 使用免疫化學染色法測定特定離子細胞 14
3.7. 使用電子掃描顯微鏡觀察離子細胞 15
3.8. 掃描式離子選擇電極技術Scanning ion-selective electrode technique (SIET) 16
3.9. RNA的製備 17
3.10.反轉錄Reverse-transcription (RT) 18
3.11.Real-time quantitative polymerase chain reaction (qPCR) 18
3.12.統計分析 19
4. 研究結果 20
4.1. AgNPs對斑馬魚胚胎體內離子含量和酸排除的影響 20
4.2. AgNPs對斑馬魚胚胎離子細胞數量和型態的影響 20
4.3. CuNPs對斑馬魚胚胎體內離子含量和酸排除的影響 22
4.4. CuNPs對斑馬魚胚胎離子細胞數量和型態的影響 22
4.5. CuNPs對斑馬魚胚胎mRNA表現量的影響 23
5. 討論 25
5.1. 奈米銀顆粒的暴露,造成斑馬魚胚胎離子調控的傷害 25
5.2. 奈米銅顆粒的暴露,造成斑馬魚胚胎離子調控的傷害 30
6. 結論 37
參考資料 38
表 1 55
圖 1~15 56
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