菟葵毒 (palytoxin; PTX) 及其類似物 (PTX and analogs; PTXs) 為毒性極高之海洋生物毒素，存在於部分菟葵物種、渦鞭毛藻及藍藻中，亦可經由食物鏈累積於生物體內，因此食用到含有這種毒素之水產品則具有中毒之風險。台灣地處副熱帶海域，亦含有毒素之生產者，且曾發生致死案例，故應正視該毒素可能對人體造成之危害，並建立相關毒性資訊。本研究將採自基隆市大坪海岸之各月份菟葵樣品以直接定序法分析其基因序列，確認所蒐集之樣品皆為同一物種 Palythoa tuberculosa。以小鼠生物檢測法 (mouse bioassay) 檢測全年之毒性，結果顯示以 8 月份及 10 月份之樣品萃取液注射小鼠後，出現後肢伸展、腹瀉及食慾不振之症狀。此外，萃取液會引起小鼠血漿中的肌酸磷酸激酶 (creatine phosphokinase; CPK) 和乳酸脫氫酶 (lactate dehydrogenase; LDH) 活性提升，且具有溶血活性，其中 8 月份及 10 月份萃取液之溶血活性分別為 10.00% 及 25.69%。由上述三項生物活性指標顯示基隆產菟葵 P. tuberculosa 之萃取液中含有 PTXs，其毒性以 8 月份及 10 月份較強。另為知 PTXs 對於血管內皮細胞 (SVEC 4-10) 之毒性作用，乃以另一批綠島產菟葵 P. tuberculosa 之萃取液進行細胞毒性試驗。MTT 試驗結果顯示萃取液 (1-25 µg/mL) 對於血管內皮細胞具有毒性，可改變細胞型態由多角狀轉變為圓球狀，然而細胞經 ouabain 預處理後，此毒性作用則會受到抑制，進一步表明萃取液中含有 PTXs 的存在。於本實驗中萃取液會造成細胞內活性氧 (reactive oxygen species; ROS) 和ㄧ氧化氮 (nitric oxide; NO) 之生成量提升，總硫醇基含量的降低，並引發粒線體膜電位下降等不利影響。採用流式細胞儀以 Annexin V/PI 雙染的方式檢測細胞死亡之途徑，可發現依萃取液濃度的不同，凋亡 (低濃度) 及壞死 (高濃度) 皆為造成細胞死亡的可能形式。綜合上述結果推測 PTXs 可能藉由使 SVEC4-10 細胞內氧化壓力提升，造成粒線體功能障礙而誘導細胞死亡。

Palytoxin (PTX) and analogs (PTXs) are a group of the most potent marine toxins produced by some zoanthids of the genus Palythoa and Zoanthus, dinoflagelllate Ostreopsis and cyanobacteria Trichodesmium. They can be also accumulated in marine organisms via the food chain. People who consume these contaiminated seafood have the risk of food poisoning. Taiwan is located in the subtropical area and has the producer of toxin.There also has been reported one case of human intoxication resulting a fatal outcome. Therefore, we should faced up to the hazard that the toxin may be harmful to human and tried to establish the relevant toxicity information. In this study, the gene sequences of zoanthid samples which collected from the Da-Ping coast of Keelung, Taiwan were analyzed by polymerase chain reaction (PCR) technique with direct sequencing. All of the samples were identified as the same species, Palythoa tuberculosa. Then the toxicity of the samples in each month was first detected by mouse bioassay. After the mice were injected with the extracts of samples collected in August and October, the mice showed PTXs symptoms such as stretching of hind limbs, diarrhea, and loss of appetite. In addition, the extracts also have the ability to increase the creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activities in mouse serum. On the other hand, hemolytic activity was detected in the extracts in August and October, which were 10.00% and 25.69%, respectively. The biological activities of above three indicators showed that the extracts of P. tuberculosa from Keelung,Taiwan contained PTXs, and its toxicity was more stronger in August and October. In order to understand the toxic effects of PTXs on vascular endothelial cells (SVEC 4-10), another zoanthid sample (P. tuberculosa) collected from Green Island,Taiwain were used for cytotoxicity test. The results of MTT assay showed that the extract (1-25 μg/mL) is toxic to SVEC4-10 cells and can change the cell morphology from polygonal to spherical. However, the cytotoxicity was inhibited after cells pretreated with ouabain. It was further suggested that the extract contained PTXs. In this experiment, the extracts could induce several adverse effects such as elevating generation of reactive oxygen species (ROS) and nitric oxide (NO), decreasing total thiol content, and reducing mitochondrial membrane potential. The type of cell death induced by the extract was investigated by Annexin V/PI double staining using flow cytometry. It was found that apoptosis (low concentration) and necrosis (high concentration) were all possible forms of cell death depending on the concentration of the extract. These results suggest that PTXs may cause increased intracellular oxidative stress, lead to mitochondrial dysfunction and finally induce cell death.

目錄

中文摘要...............................................I
Abstract..............................................II
壹、文獻整理........................................... 1
一、菟葵之簡介......................................... 2
二、菟葵毒及其類似物之簡介.............................. 3
三、菟葵毒及其類似物之檢驗方法............................7
四、以分子生物技術鑑定物種...............................10
貳、研究內容........................................... 11
第一章、基隆產菟葵 Palythoa tuberculosa 之季節毒性分析....12
一、前言................................................12
二、材料與方法...........................................13
三、結果................................................18
四、討論................................................20
五、圖表................................................22
第二章、菟葵毒及其類似物對於血管內皮細胞之毒性探討..........33
一、前言................................................33
二、材料與方法...........................................34
三、結果................................................40
四、討論................................................42
五、圖表................................................45
參、總結論.............................................. 56
肆、參考文獻.............................................57
伍、附錄.................................................66
謝辭.....................................................69

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