砷(Arsenic，As)是以化合物型態普遍存在於自然界中的類金屬物質。無論是自然或人為因素來源，在台灣曾多次引起公共衛生問題。生物體在攝入砷以後會導致生理機能上的異常，極低濃度下促使細胞增生，高濃度下抑制生物代謝並引發多重器官病變。一般而言無機砷毒性遠大於有機砷，攝入無機砷後的解毒路徑是經由甲基化作用轉化成有機砷代謝出體外。
    本論文的實驗以斑馬魚作為生物模式(animal model)，以溶劑萃取代謝物並冷凍乾燥後，利用NMR為分析工具，判定並定量各代謝物質。觀察給予不同砷環境下，對斑馬魚代謝上的探討。
    實驗發現斑馬魚因砷而增強甲基化代謝能力，且TMAO能作為斑馬魚健康狀況的指標性代謝物，並証實斑馬魚與NMR的組合能提供研究代謝變化的可行方式。

Arsenic is common compounds in nature, and caused many public health problems in Taiwan. Organisms take in arsenic lead to abnormal physiological function. Low concentrations promote cell proliferation. High concentrations inhibit the metabolism and cause multiple organ disease. Generally, inorganic arsenic is usually more toxic than organic arsenic. Organisms take in inorganic arsenic the detoxification pathway is through the methylation into organic arsenic.
    The zebrafish is an animal model in this thesis. Use solvent to extraction and freeze-dried the zebrafish product. Analyse metabolites include determination and quantification by NMR. Observe different arsenic conditions the metabolism research of zebrafish.
    Experiment was found the zebrafish is enhanced methylation metabolism by arsenic. TMAO can as indicator metabolite of health status in zebrafish. Confirmed the combination of zebrafish and NMR provide a viable way of metabolic study.

目錄   
目錄	I
表目錄	IV
圖目錄	V
縮寫表	IX

第一章 緒論	1
1-1砷的來源	1
1-1.1 自然界之來源	1
1-1.2 人為之來源	2
1-2 砷對生物的影響	2
1-2.1 砷中毒的症狀	3
1-2.2 生物的抗砷機制	4
1-3 試驗生物斑馬魚(zebrafish, Denio rerio)介紹	7
1-4 實驗儀器核磁共振儀(NMR)基本介紹	9
1-5 實驗動機及目的	11
第二章 原理	12
2-1.1 NMR的基本原理	12
2-1.2 化學位移 (Chemical Shift)	13
2-2 二維核磁共振	15
2-2.1 COSY光譜(Correlation Spectroscopy)	17
第三章 實驗材料與方法	20
3-1 實驗用斑馬魚(Danio rerio)之飼養	20
3-2 砷化物的浸泡實驗	20
3-2.1 半數致死濃度	21
3-2.2 不同濃度之砷浸泡	21
3-2.3 砷化物對不同週期之斑馬魚體內代謝影響	21
3-3 斑馬魚之萃取與配製NMR tube	25
3-4 NMR實驗操作	27
3-5 數據計算	29
第四章 實驗結果	30
4-1 半數致死濃度實驗存活率	30
4-2 As-treated III 抗(耐)砷實驗存活率	31
4-3 1D光譜判定(有機相)	34
4-4 1D 光譜判定(水相)	37
4-5 COSY光譜判定 (assignment)	42
4-6 斑馬魚Mock組定量	56
4-7 斑馬魚As-treated I定量	60
4-8 斑馬魚As-treated II定量	63
4-9 斑馬魚As-treated III定量	66
4-10 斑馬魚水相代謝物As濃度組定量	69
第五章 討論	72
5-1 Mock組中斑馬魚的代謝變化討論	72
5-2 As濃度對斑馬魚造成的影響	73
5.3 各種As-treated對斑馬魚造成的影響	74
5-3.1. As-treated I探討	74
5-3.2. As-treated II探討	76
5-3.3. As-treated III探討	77
第六章 結論與未來展望	81
參考資料	83
               













表目錄
表4-1 半數致死濃度實驗存活統計表	30
表4-2 抗(耐)砷實驗存活統計表(24小時紀錄)	33
表4-3 抗(耐)砷實驗存活統計表(每天記錄)	34
表4-4 NMR有機相光譜Assignment	36
表4-5 NMR水相光譜Assignment	40
表4-6 水相代謝物Mock組定量總表	57
表4-7 As-treated I定量總表	61
表4-8 As-treated II 定量總表	64
表4-9 As-treated III 定量總表	67
表4-10 斑馬魚水相代謝物As濃度組定量總表	70
                        















圖目錄
圖1-1 砷在人體內經由甲基化代謝的解毒途徑	6
圖2-1 鮭魚肌肉水相萃取物之1D氫譜代謝物質分布圖	14
圖2-2 二維核磁共振實驗的脈衝序列	15
圖2-3 在二維核磁共振實驗中，將t1 做線性增加	16
圖2-4 COSY 的脈衝序列	17
圖3-1砷化物對不同週期之斑馬魚體內代謝影響實驗示意	24
圖4-1 半數致死濃度實驗存活率趨勢圖	31
圖4-2.1 抗(耐)砷實驗存活率狀況(24小時)	32
圖4-2.2 抗(耐)砷實驗存活率狀況(每天記錄)	32
圖4-3.1 有機相1D NMR光譜	35
圖4-3.1 有機相1D NMR光譜	35
圖4-4.1 水相1D NMR光譜	38
圖4-4.2 水相1D NMR光譜	38
圖4-5.1 LEU 2D COSY 判定	43
圖4-5.2 ILE 2D COSY 判定	44
圖4-5.3 VAL 2D COSY 判定	45
圖4-5.4 LA 2D COSY 判定	46
圖4-5.5 ALA 2D COSY 判定	47
圖4-5.6 LYS 2D COSY 判定	48
圖4-5.7 GLU 2D COSY 判定	49
圖4-5.8 GLN 2D COSY 判定	50
圖4-5.9 MA 2D COSY 判定	51
圖4-5.10 CR/PCR 2D COSY 判定	52
圖4-5.11 TAU 2D COSY 判定	53
圖4-5.12 GB 2D COSY 判定	54
圖4-5.13 a-GLC 2D COSY 判定	55
圖4-6.1圖4-6.1 amino acid 比較圖 ..………………………………....59
圖4-6.1圖4-6.1 amino acid 比較圖 ..…………………………………59
圖4-7 As-treated I與Mock組的代謝濃度差異比較	62
圖4-8 As-treated II與Mock組的代謝濃度差異比較	65
圖4-9 As-treated III與Mock組的代謝濃度差異比較	68
圖4-10 As濃度組與Mock組的代謝濃度差異比較	71

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