臺灣博碩士論文加值系統

河魨毒 (tetrodotoxin; TTX) 是一種具有強烈毒性的非蛋白毒素，可藉由食物鏈累積的方式存在於生物中，因此 TTX 除河魨外，也見於蝦虎魚、螃蟹及扁蟲等其他物種。國內每年均有食用含 TTX 所引發之食物中毒案例發生，近年來因氣候變遷，以及「萊塞普遷徙」(Lessepsian migration) 的緣故，近幾年歐洲國家陸續出現河魨毒中毒案例。中毒患者症狀包括口腔感覺異常、嘔吐、腹瀉、呼吸困難甚至死亡，為確保食品安全管控，各物種河魨之毒性及毒素分布資料應予以建立分析。另外，由於 TTX 具有阻塞鈉離子通道使動作電位無法產生之特性，近年來嘗試被開發成抗痛藥劑，因此不同細胞暴露於 TTX 後所引發之凋亡路徑及細胞毒性也應被確立，以助於日後做為醫療用途參考。
本研究第一章擬自漁市捕獲未知種類之有毒河魨，以及本實驗室接獲河魨毒中毒案件之另件河魨樣品，進行物種鑑定及毒性分析。兩種樣品經基因分析鑑定分別確認為四齒魨科魚種星斑叉鼻魨 Arothron stellates 及紋腹叉鼻魨 Arothron hispidus。為了解此兩品種河魨體內毒素的含量與分布，乃萃取不同部位之毒素，利用 C18 Sep-Pak cartridge column 進行純化，得到較高純度之 TTX，然後以生物試驗法及 LC-HRMS 法做毒素之定量與定性以及毒成分分析。結果顯示兩種河魨中皆含有數種 TTX 衍生物，星斑叉鼻魨以卵巢含最高毒性 (257.39 µg/g)，紋腹叉鼻魨則以肌肉最毒 (1.25 µg/g)，以生物檢驗法與 LC-HRMS 檢驗法定量結果之比值介於 60-150% 之間，說明兩方法毒素定量結果相似。
第二章研究則延續前一章之方法，自星斑叉鼻魨肝臟萃取出毒素後純化，並以此進行 TTX 對於小鼠海馬神經元細胞 (mouse hippocampal neuronal cell line) HT-22 細胞毒性作用之探討。將細胞暴露於 TTX 後，測定其氧化壓力、粒線體損傷情形，除了凋亡前現象進行個別檢測外，亦測定細胞凋亡壞死分析。結果顯示，細胞於各濃度培養三天後，存活率成濃度效應，其胞內氧化壓力隨濃度上升而增加，而粒線體膜電位隨濃度增加而下降。最後以 Annexin-V/PI 染劑檢測，發現在低濃度毒素作用時，細胞多呈現凋亡，推論 TTX 將造成細胞走向傷害粒線體相關途徑之細胞凋亡，而以高濃度作用時，細胞壞死的比例增加。

註：萊塞普遷徙 (Lessepsian migration) 指蘇伊士運河上的海洋物種遷徙，通常是由紅海到地中海，相反方向則較少。

Tetrodotoxin (TTX) is a non-protein toxin with strong toxicity. It can major exist in organisms through accumulation of food chains. TTX is also found in other species such as goby, crab, flatworm and so on. There are several cases of food poisoning caused by food containing TTX every year in Taiwan. In recent years, due to climate change and the "Lessepsian migration", cases of pufferfish poisoning also happened in European countries. Symptoms of poisoning include oral paresthesia, vomiting, diarrhea, dyspnea and even death. To ensure food safety and contol, the toxicity and toxin distribution data of new pufferfish should be perfectly established. In addition, since TTX has the property of blocking sodium channels and preventing action potentials from being generated, it has been attempted to be developed as an anti-pain agent in recent years. Therefore, the apoptosis pathway and cytotoxicity induced by TTX should be determined for medical purposes in the future.
The first chapter of this study intends to identify the species and analyze the toxicity of the pufferfish from the market and the poisoning cases. Two pufferfishes were sparately identified as tetraodontidae Arothron stellatus and Arothron hispidus. In order to understand the content and distribution of toxins in these species, the toxins from different organs were extracted. The C18 Sep-Pak cartridge column was used for purification and TTX with higher purity was obtained. Then the quantitative and qualitative analysis of the toxins were carried out by bioassay and LC-MS/MS method. The results showed that there were several analogues of TTX in both species. The highest toxicity (257.39 μg/g) was found in the ovaries of Arothron stellatus and the most toxic organ is muscle (1.25 μg/g) in Arothron hispidus. The ratio of the quantitative results of the bioassay to the LC-MS/MS test ranged between 60 and 150%, indicating that the quantitative results of the two methods were similar.
The second chapter of the study followed the method of the previous chapter. After the toxin extracted from the liver of Arothron stellatus, it was purified and treated to mouse hippocampal neuron cell, HT-22 cells, to investigate the toxicological effects. The results showed that toxins could induce reactive oxygen species (ROS) and cause oxidative stress after the cells were cultured for three days at each concentration. Meanwhile, mitochondrial membrane potential (MMP) decreases with dose-dependent and finally causes cell apoptosis. Finally It was found that cells exhibited apoptosis at low concentrations of toxin, whereas the proportion of cell necrosis increased at high concentrations.

中文摘要 I
Abstract II
目錄 1
壹、文獻整理 3
一、河魨毒之一般特性 4
(一) 化學性質 4
(二) 毒作用機制 4
(三) 自然界之來源與分布 5
(四) 中毒症狀 6
(五) 臨床治療 7
(六) 醫療上之應用 7
二、河魨毒之檢測方法 9
(一) 生物檢定法 (Bioassay) 9
(二) 薄層色層分析法 (Thin-layer chromatography, TLC) 9
(三) 電泳分析 (Electrophoresis) 9
(四) 高效能液相層析法 (High performance liquid chromatography; HPLC) 9
(五) 液相層析質譜法 (Liquid chromatography mass spectrometry; LC-MS） 10
(六) 液相層析串聯式質譜法 (Liquid chromatography tandem mass spectroscopy analysis; LC-MS/MS) 10
(七) 免疫分析法 (Immunoassay) 10
(八) 組織培養生物檢定法 (Tissue culture bioassay) 10
三、中毒案例 11
(一) 國內案例 11
(二) 國外案例 12
貳、研究內容 13
第一章、臺灣產叉鼻魨屬河魨毒性毒成分分析 14
一、前言 14
二、材料及方法 15
三、結果 21
四、討論 24
五、圖表 26
第二章、河魨毒對於 HT-22 神經細胞毒性之探討 58
一、前言 58
二、材料與方法 59
三、結果 64
四、討論 66
五、圖表 68
參、總結論 75
肆、參考文獻 77
謝辭 85

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