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研究生:李念蓁
研究生(外文):Nian-JhenLi
論文名稱:奈米銀對斑馬魚不同胚胎發育階段之毒性效應及其在建構物質安全資料表的可能貢獻
論文名稱(外文):The toxic effects of silver nanoparticles on developmental-specific embryonic phenotypes in zebrafish and its possible application in material safety data sheets
指導教授:王應然王應然引用關係
指導教授(外文):Ying-Jan Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境醫學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:89
中文關鍵詞:斑馬魚2-氨基乙硫醇奈米銀微粒奈米毒性物質安全資料表
外文關鍵詞:zebrafishcysteamine-modified silver nanoparticleNanotoxicitymaterial safety data sheet
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奈米銀微粒具有特殊的物理化學特性,例如導電性好、穩定性佳且具有抗菌能力等特性,因此奈米銀微粒從先進的科技到醫學技術及民生用品皆被廣泛的應用,但同時也增加環境及人體暴露到奈米銀微粒的機會。儘管奈米銀微粒被大量地使用,但其對生物體的毒性效應資訊仍然相當缺乏,也因此奈米材料的物質安全資料表(SDSs)無法完整的被建立。斑馬魚具有飼養成本低、短時間內可大量繁殖、胚胎透明、胚胎發育時間快等優點,因此被認為是一個良好的動物模式,國際標準組織(International Organization for Standardization;ISO)也將斑馬魚列為奈米毒性測試的建議動物模式,而歐洲經濟合作暨發展組織 (Organisation for Economic Co-operation and Development; OECD)也已經建立數個使用斑馬魚作為毒性測試之指引。因此本研究利用斑馬魚模式探討以2-氨基乙硫醇修飾之大粒徑(Large size cysteamine-modified silver nanoparticle; LAS)與小粒徑(Small size cysteamine-modified silver nanoparticle; SAS)奈米銀微粒對斑馬魚不同胚胎發育階段之毒性效應,並且蒐集、整理及歸納現有的奈米銀微粒物質安全資料表,將可行的實驗數據應用於安全資料表的毒性資料或生態資料之項目。本研究先製備LAS (55.6±13.8 nm)及SAS (30.0±7.3 nm),並測定其物化特性。將斑馬魚胚胎暴露於製備的奈米銀溶液中,於28℃培養至不同的發育時期,每日更換暴露溶液並觀察記錄存活率與畸形狀態,於試驗終點分析包含氧化壓力等毒性效應。本研究也使用Tg(LFABP:EGFP)肝臟基因轉殖斑馬魚、Tg(IFABP:dsRed)腸道基因轉殖斑馬魚以及huORFZ基因轉殖斑馬魚,分析奈米銀微粒造成的肝臟、腸道之發育影響及內質網壓力。此外,本研究也利用網路搜尋的方式,收集由供應商或製造商提供的19件奈米銀微粒物質安全資料表,並且進行內容的分析及整合。由時序性存活率(time-course survivorship)毒性試驗結果指出,斑馬魚胚胎分別暴露LAS及SAS後,其胚胎存活率皆隨著濃度增加而下降,存在劑量效應關係,並且LAS之毒性高於SAS。由胚胎存活率試驗結果也發現,斑馬魚胚胎發育的早期階段比後期階段對於奈米銀微粒具有較高的敏感性。奈米銀微粒亦會引起斑馬魚胚胎發育的異常,包括卵黃的不透明或水腫、心包膜水腫、體軸彎曲、以及胚胎發育延遲。利用螢光標記的奈米銀微粒測試後發現,LAS及SAS皆會穿過卵膜進入胚胎,而LAS進入胚胎之含量較SAS多。另外,奈米銀微粒會在胚胎的腸道及肝臟誘發氧化壓力,並造成肝細胞與腸道細胞受損。奈米銀微粒也會使胚胎產生內質網壓力,暴露LAS的胚胎其內質網壓力會表現在肝臟及腸道,而暴露SAS的胚胎則表現在卵黃囊的表面。而奈米銀微粒也會造成溶酶體的活性增加,可能也會誘發自體吞噬的作用。本研究也觀察到有凋亡細胞的產生,可能是造成胚胎畸形或發育遲緩的原因之一。在奈米物質安全資料表中,大多數奈米銀SDSs的內容並未包含充分的奈米材料安全資訊,例如奈米材料的毒性和物化特性,因此我們根據 ISO_TR_13014及13329(2012)之技術指南,將蒐集之文獻以及本研究分析結果,包含前述的存活率毒性試驗結果計算所得之該奈米物質的LD50及EC50,填入物化特性、毒理及生態毒性資訊的項目,提供初步的奈米銀SDSs模板及相關資訊。
Due to nanoparticles have unique physical and chemical properties, nanotechnology is rapidly growing with nanoparticles produced and utilized in a wide range of commercial products throughout the world. Silver nanoparticles (AgNPs) are the most commonly used among nanomaterials, which is a consequence of the above-noted antimicrobial properties of Ag. AgNPs are extensively used in electronics, catalysts, biosensors, medical devices, cosmetics and large commercial antibacterial products. These wide applications increase human and environment exposure and thus the potential risk related to their short- and long-term toxicity. The purpose of this study is to investigate the toxic effects and mechanism of AgNPs on zebrafish embryos. The results indicated zebrafish embryos exposed with AgNPs lead to reduce the survival ratios in a size-, dose-, and time dependent manner. The embryos treated with the AgNPs develop into abnormal phenotypes (abnormal jaw, axis flexure, pericardial edema, and yolk sac edema) and developmental retard. Our results implied that AgNPs-induced toxicity include oxidative stress, ER stress and apoptosis by particle can uptake into the embryos via chorion. These results show that different size of the AgNPs can trigger different toxicity, the large size cysteaamine-modified AgNPs were more toxic than small size cysteaamine-modified AgNPs. By comparing the safety material data sheets (SDSs) information between silver nano-sized and silver bulk-sized materials and performing current knowledge integration, we can provide more valid information on hazard identification and toxicological information of nano-safety data sheets.
第一章、序論 1
第二章、文獻回顧 2
第一節、奈米材料之應用 2
第二節、奈米材料物化特性及其潛在生物效應 2
第三節、奈米銀微粒之應用及表面修飾的效應 3
第四節、奈米銀微粒於in vitro與in vivo之毒性效應 4
第五節、奈米微粒生物危害評估動物(體內)模式 — 斑馬魚 6
第六節、奈米微粒之毒性機制與重要生物指標 13
第七節、奈米微粒物質安全資料表(MSDS)之發展需求與建立 17
第三章、研究目的 20
第四章、研究材料與方法 21
第一節、研究材料 21
第二節、研究方法與實驗步驟 25
一、奈米銀微粒合成 25
二、奈米銀微粒之物化特性分析方法 26
三、斑馬魚飼養 (Zebrafish husbandry) 27
四、斑馬魚配對方法 (Breeding of zebrafish embryos) 28
五、斑馬魚胚胎毒性試驗 28
六、斑馬魚胚胎毒性試驗生物指標 29
七、氧化壓力(Oxidative Stress)分析 29
八、以huORFZ基因轉殖斑馬魚分析奈米銀在斑馬魚誘發的內質網壓力 30
九、以Tg(LFABP:EGFP)肝臟基因轉殖斑馬魚及Tg(IFABP:dsRed)腸道基因轉殖斑馬魚評估奈米銀對標的器官的發育影響 30
十、細胞凋亡試驗-Whole-mount TUNEL assay 30
十一、斑馬魚胚胎之奈米銀微粒分布 30
十二、斑馬魚胚胎內之銀離子含量 31
十三、溶酶體活性(Lysosomal activity)分析 32
十四、免疫螢光染色(Whole-mount immunostaining) 32
十五、西方墨點法(Western Blot)分析 33
十六、統計分析 34
第五章、研究架構 35
第六章、研究結果 36
第一節、奈米銀微粒物化特性分析 36
第二節、2-氨基乙硫醇奈米銀微粒對於斑馬魚胚胎之毒性結果 36
第三節、斑馬魚胚胎暴露奈米銀微粒之外觀變化與畸形反應 37
第四節、暴露奈米銀微粒影響斑馬魚胚胎的發育 37
第五節、暴露奈米銀微粒後進行氧化壓力試驗 38
第六節、暴露奈米銀微粒後胚胎標的器官的影響 39
第七節、斑馬魚胚胎暴露奈米銀微粒之分布情形 39
第八節、斑馬魚胚胎暴露奈米銀微粒之銀離子含量測定 40
第九節、以huORFZ基因轉殖斑馬魚分析奈米銀在斑馬魚誘發的內質網壓力 40
第十節、暴露奈米銀微粒後以LysoTracker進行溶酶體活性的偵測 40
第十一節、暴露奈米銀微粒後細胞凋亡分析 40
第十二節、暴露奈米銀微粒後誘發細胞自體吞噬與內質網壓力之相關蛋白表現 41
第七章、討論 42
第八章、結論及建議 47
第九章、參考文獻 48
圖表 56


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