D型胺基酸氧化酶(D-amino acid oxidase；DAO)是一種含有FAD輔因子的氧化酵素，廣泛的存在微生物與動物組織內。早期DAO的研究大多著重於活體外的酵素活性分析，製造大量的DAO酵素，應用於抗生素製造與胺基酸代謝失常病人的治療，近年來關於DAO研究開始著重於活體內的DAO分布與相關的生理功能做深入的探討。而苯甲酸鈉(Sodium Benzoate；SB)是DAO的競爭型抑制劑，苯甲酸鈉為一種廣泛使用在化妝品與食品上的防腐劑，可用來治療尿素循環失調的病患；但是SB在胚胎發育早期的潛在危險性仍然未知。本論文以斑馬魚(Danio rerio, zebrafish)當作實驗材料，藉由斑馬魚的產卵率高、胚胎透明且可觀察器官形成等優點來探討斑馬魚DAO之酵素活性分析與苯甲酸鈉在胚胎發育早期所引發的潛在毒害現象。本論文的第一部分，我們複殖出斑馬魚DAO基因，並進行定序與各物種間的比對，發現斑馬魚DAO胺基酸序列與鯉魚有93%的相似度；以大腸桿菌表現系統進行N端含有His．Tag的斑馬魚DAO表現蛋白之誘導與純化，測得斑馬魚DAO酵素比活性為0.40±0.27 U/mg，接著測定不同時期之斑馬魚DAO酵素比活性，我們觀察到斑馬魚體內的DAO酵素比活性在三天大時為0.148±0.025 U/mg，接著陸續增加至六天大時達到最高值0.323±0.040 U/mg，然後活性逐漸下降至成體時為0.019±0.005 U/mg。我們純化並測得斑馬魚DAO表現蛋白的活性，且發現在不同時期的斑馬魚胚胎體內有不同含量的DAO酵素存在。在本論文的第二部分，我們利用不同濃度的苯甲酸鈉來浸泡斑馬魚胚胎，發現在低於1000 ppm的SB浸泡下，斑馬魚有100%的存活率，隨著苯甲酸鈉濃度的增加存活率也逐漸下降，至2000 ppm的苯甲酸鈉浸泡下，斑馬魚全部死亡。浸泡過苯甲酸鈉的斑馬魚其外觀上有微心腔(pericardial sac)腫大、孵化腺體(hatching gland)與腸道(gut)等之變異，隨著苯甲酸鈉濃度的降低而外觀上的變異情形隨著減輕。接著利用神經與肌肉專一性抗體進行免疫螢光染色，我們發現苯甲酸鈉會造成斑馬魚胚胎的側線神經節消失、RB (Rohon-Beard neuron)神經細胞死亡延遲、分支出許多的運動神經軸突和運動神經肌肉接點與慢肌纖維排序混亂等現象，但肌肉的α-actin mRNA沒有受到苯甲酸鈉浸泡的影響。綜合以上結果，我們認為苯甲酸鈉在斑馬魚胚胎發育早期除了會導致感覺神經的毒害現象，也會造成肌肉纖維排序混亂，進而使運動神經元無法與目標肌肉相接，而形成許多分支的運動神經肌肉接點，間接的影響到釋放神經傳導物質的運動神經軸突前突觸端。

D-amino acid oxidase (DAO) is a FAD-containing enzyme which has been characterized in microorganisms and animals. On early periods, the studies of DAO were focused on analysis of enzymatic activity, large-scale production of DAO enzyme, to produce antibiotic and in treatment of patients who had amino acid metabolism disorders. Recently, the distribution and physiological functions of DAOs have become an important issue to be addressed. Sodium benzoate (SB) is a competitive inhibitor of DAO in vitro. SB is a widespreadly used preservative on cosmetic industry and food. It was also used in the treatment of hyperammonaemia in patients with inborn errors of the urea cycle. However, little is unknown about whether SB possessed potential toxicities during early development. Here, we used zebrafish as a model to characterize the enzymatic activity analysis of zebrafish DAO (ZDAO) and to test SB-induced toxicity during early embryonic development of zebrafish. We chose zebrafish as our animal model for the following reasons: large amount and transparent eggs, easy to observe during organogenesis. In the first part of this thesis, we cloned and sequenced a ZDAO gene. In terms of amino acid sequence comparison, ZDAO shared 93% identity with the carp’s DAO. A 39 kDa 6xHis-ZDAO fusion protein was induced and purified in Escherichia coli expression system. The specific enzymatic activity of 6xHis-ZDAO fusion protein was 0.40±0.27 U/mg. The enzymatic specific activities of  endogenous ZDAO at different developmental stages were also measured. The ZDAO enzymatic specific activities at 3dpf was 0.148±0.025 U/mg, reached to the optimal at 6dpf (0.323±0.040 U/mg) and gradually decreased to 0.019±0.005 U/mg at adult fish. These results indicated that ZDAO expression profile was dynamics. In the second part of this thesis, we treated zebrafish with different concentrations of SB. After low dosage SB (1~1000 ppm) treatment, the zebrafish embryos exhibited 100% survival rates. As the exposure dosages increased, the survival rates decreased. No embryos were survival after treatment with 2000 ppm of SB. Morphological defects were observed, including hatching gland abnormalities, gut abnormalities and edema in pericardial sac. In addition to the predominant morphological defects in gut and heart, we also found that SB-treated embryos had the following defects that revealed by using antibodies against mature neurons (anti-acetylated-tubulin), sensory neurons (Zn12), motor neurons (Znp1 and Zn5), neuromuscular junctions (Znp1 and α-BTX) and muscle fibers (F59), including: disturbed muscle fibers, missing of neuromast, prolonged RB (Rohon-Beard) neuron lifespan and dispersed trunk motoneuron projections and neuromuscular junctions. Based on these observations, we conclude that SB is able to induce toxicities on sensory neurons, muscle fibers, motor neurons and neuromuscular junctions during early embryonic development of zebrafish.

中文摘要…………………………………………………………………………………I
Abstract………………………………………………………………………...………III
目   錄………………………………………………………………………………….V
圖、表 目錄……………………………………………………………………………VI
第一部份：斑馬魚D型胺基酸氧化酶（D-Amino Acid Oxidase）之基因表現與酵素活性分析
一、	前言……………………………………………………………………………2
二、	材料與方法……………………………………………………………………8
三、	結果………………………………………………………………………...…18
四、	討論…………………………………………………………………………...25
五、	圖、表……………………………………………………………………...…33
第二部份：苯甲酸鈉（Sodium Benzoate）在斑馬魚胚胎發育早期之毒性測試
一、	前言……………………………………………………………………..….…46
二、	材料與方法……………………………………………………………….…..50
三、	結果………………………………………………………………………...…56
四、	討論…………………………………………………………………………...63
五、	圖、表………………………………………………..……………………….71
參考文獻…………………………………………………………………….………..…87
附錄…………………………………………………………………………………….101
第一部份圖、表
圖1：ZDAO之核苷酸與胺基酸序列…………………………..………………...33
圖2：各物種之DAO胺基酸一級結構之比對……………………..……………34
圖3：DAO胺基酸序列同源性演化樹狀圖……………………………..……….35
圖4：pET28c-DAO表現質體之架構…………………………………………….36
圖5：37℃下乳糖誘導ZDAO表現蛋白的結果…………………………………37
圖6：低溫誘導下的ZDAO表現蛋白之分佈情形………………………………38
圖7：加入D-Sorbitol與Betaine誘導ZDAO表現蛋白之影響…………….…..39
圖8：Inclusion body ZDAO表現蛋白之尿素溶解與純化……...……………….40
圖9：Inclusion body ZDAO表現蛋白之西方墨漬法………….……………..….41
圖10：可溶性ZDAO表現蛋白之純化…………………………………………..42
圖11：不同時期之ZDAO酵素比活性測定……………………………………..43
表1：各物種之DAO胺基酸序列相似度比較表….…………………………….44
表2：ZDAO表現蛋白之活性與比活性測定……………………..……………...44

第二部份圖、表
圖1：斑馬魚胚胎於不同濃度的SB浸泡下之存活率…………………………..71
圖2：斑馬魚胚胎於SB浸泡下的外觀變異情形………………………………..72
圖3：斑馬魚胚胎於1000 ppm SB浸泡下之存活率…………………………….73
圖4：斑馬魚胚胎之成熟神經染色……………………………………………….74
圖5：斑馬魚胚胎之RB神經細胞染色……………………………………....…..76
圖6：斑馬魚胚胎之死亡的RB神經細胞染色…………………………………..77
圖7：斑馬魚胚胎之運動神經軸突投射分布情形…………………………...……78
圖8：斑馬魚胚胎之二級運動神經軸突分布情形…………………………….....79
圖9：斑馬魚胚胎之運動神經軸突與運動神經肌肉接點分布情形…………….80
圖10：斑馬魚胚胎之慢肌纖維分布情形………………………………………....81
圖11：18小時的斑馬魚胚胎之肌肉α-actin mRNA分布………………….…...82
圖12：三天大的斑馬魚胚胎之肌肉α-actin mRNA分布…………………….....83
圖13：斑馬魚胚胎之運動神經肌肉接點與肌肉的排列分布圖……………..….84
表1：浸泡過SB的三天大斑馬魚胚胎之外觀變異情形……………………..…85
表2：浸泡過SB的兩天大斑馬魚胚胎之成熟神經染色的變異情形………..…86

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