咖啡因是一種廣泛的存在於許多食品中，包括咖啡、茶葉、可可。然而，在我們食用或飲用這些日常食品的時候，常常不自覺得過量食用咖啡因。咖啡因的結構為一種生物鹼，這種物質對人類而言是一種植物性的中樞神經興奮劑 (O'Connell and Zurzola, 1984)，對於研究咖啡因在人體中的作用已經有許多的報導。在服用50-200mg的咖啡因後，會產生潮紅、寒顫、煩亂、興奮、沒有胃口、虛弱、顫抖，另外還有高血壓、低血壓、心跳過速、嘔吐、發熱、妄想症、幻覺、疾病發作、心律不整、心臟停止跳動、昏迷和死亡(Kerrigan and Lindsey, 2005)；若是口服大量的劑量（服用超過5 g的咖啡因）是一種極度有毒的藥劑，會造成人體的心律不整，心跳過快、嘔吐、抽搐、昏睡和死亡 (Kerrigan and Lindsey, 2005)。但是我們仍然對咖啡因在胚胎發育時期的影響並不明瞭，所以我們接著選擇斑馬魚作為我們的模式物種，以斑馬魚為模式物種的優點有：個體數高和胚胎透明容易觀察。這兩個優點讓我們能夠更細微地觀察咖啡因在斑馬魚早期發育所造成的影響。因此我們找到咖啡因對斑馬魚胚胎的半數致死濃度，LC50為250 ppm。然而，我們為了收集更多的觀察樣本，於是將濃度調降為150 ppm，並觀察其外觀變異，發現胚胎的體長變短。接著在能動性 (motility) 的測驗下，咖啡因濃度越高，能動性越低。於是，我們以F59、Znp1、aat螢光抗體染色做更細微的觀察，結果發現肌纖維排列混亂，運動神經軸索（axon）無法正常延伸發育、感覺神經數量減少、腦部的發育延遲、軀體彎曲以及心臟和血管發育不良的現象。由此，我們認為咖啡因對斑馬魚胚胎對神經發育、肌肉發育、以及心臟發育具有相當的毒性。

Caffeine is a commonly existing in many kinds of food, including coffee, tea, and coca. However, little is known about the caffeine-induced toxicity and teratogenicity during early embryonic development. Here, we used zebrafish as a model to test the toxicity and teratogenicity because of their transparent eggs; therefore, the organogenesis of zebrafish embryos is easy to observe. After low dosages of caffeine (1-100 ppm) treatment, the zebrafish embryos exhibited a 100 % survival rate. As the exposure dosages increased, the survival rates decreased. No embryos survived after treatment with 1000 ppm caffeine. The 50% lethal dose (LD50) of zebrafish is found to be in the range of 200-275 ppm. Shortened and curved body were observed after treatment with caffeine. Compared to untreated littermates (vehicle-treated control), caffeine-treated embryos exhibited significantly reduced tactile sensitivity frequencies of touch-induced movement (vehicle-treated control: 10±3 v.s. 150 ppm caffeine: 0±0; N=30). Subtle changes are easily observed morphology by staining with specific monoclonal antibodies F59, Znp1 and aat to detect changes in muscle fibers, motor axons, sensory neurons, Rb cell and brain, respectively. Our data showed that the treatment of caffeine led to misalignment of muscle fibers, motor neuron innervations, less acetyl-choline receptor cluster and Rohon-beard cell. On the basis of these observations, we suggest that caffeiene is able to induce neurotoxicity and nephrotoxicity of zebrafish larvae.

目錄
第一章：前言--------------------------  1
第一節：咖啡因的來源---------------------  1
第二節：咖啡因的用途---------------------  1
第三節：咖啡因對人體的作用---------------  3
第四節：咖啡因的毒性---------------------  6
第五節：研究目的------------------------  9
第二章：材料與方法------------------- 11
第一節：咖啡因浸泡實驗------------------- 11
第二節：半數致死濃度(Median Lethal      Concentration, LC50,24hr)--------------- 11
第三節：不同濃度的咖啡因浸泡-------------- 12
第四節：斑馬魚隻飼養與胚胎收集------------ 12
第五節 ：小量質體DNA之萃取-------------- 12
第六節：合成探針------------------------ 13
第七節：Whole-mount In Situ Hybridization
-------------------------------------- 13
第八節：免疫抗體染色-------------------- 15
第九節：α-bungarotoxin (α-BTX) 與Znp1的
染色----------------------------------- 15
第十節：胚胎包埋及冷凍切片---------------  17
第十一節：能動性 (motility) 的探討-------  18
第十二節：以咖啡因長時間飼養斑馬魚的觀察--
--------------------------------------- 18
第十三節：顯微照相系統------------------  19
第三章：結果-------------------------  20
第一節：半數致死濃度 -------------------  20
第二節：咖啡因對斑馬魚胚胎所造成的外觀變異  21
第三節：咖啡因對心跳的影響--------------  22
第四節：能動性 (motility) 的探討---------  24
第五節：咖啡因影響慢肌纖維排列-----------  25
第六節：咖啡因對成熟神經的影響-----------  30
第七節：感覺神經的分佈統計---------------  31
第八節：運動神經的連結和型態-------------  32
第九節：次級運動神經的發育情形-----------  33
第十節：咖啡因對神經早期發育的轉錄調節蛋白  35
第十一節：咖啡因對3 mpf時期斑馬魚骨骼的影
響------------------------------------- 38
第四章：討論------------------------ 40
第五章：參考文獻-------------------  46
圖表--------------------------------  53
圖表目錄
圖1:斑馬魚胚胎於不同濃度的咖啡因浸泡下之存活率--- 54
圖2：斑馬魚經咖啡因浸泡後的變異情形--------------- 55
圖3：咖啡因對體長體寬的影響----------------------- 56
圖4：斑馬魚胚胎於不同濃度的咖啡因之心跳數統計圖--- 57
圖5：斑馬魚胚胎的能動性測試----------------------- 59
圖6：咖啡因影響慢肌纖維排列---------------------- 60
圖7：Tnnt1 和Tnnt3b的基因表現-------------------- 60
圖8：斑馬魚胚胎之成熟神經------------------------- 63
圖9：斑馬魚胚胎之感覺神經分布情形----------------- 65
圖10：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---------------------------------- 66
圖11：斑馬魚胚胎之次級運動神經軸突投射分布情形--- 67
圖12：斑馬魚胚胎之neurogenin 1 和neuron D 基因的表
現分布------------------------------------ 68
圖13：斑馬魚胚胎之 Nav1.6基因的表現分布-----------70
圖14：斑馬魚胚胎之Krox20基因的表現分布-----------72
圖15：以10 ppm咖啡因飼養斑馬魚至成體造成骨骼變異-73

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