台灣眼鏡蛇心臟毒素 (Cardiotoxins)是由60個胺基酸所組成的鹼性三指環狀蛋白質，其展現毒性主要作用目標是細胞膜，儘管台灣眼鏡蛇心臟毒素的六種異構物胺基酸序列高度相似，卻表現出不同的細胞毒性與溶血能力，因此本研究藉由觀察心臟毒素對不同脂質組成的脂質膜之作用，試圖瞭解心臟毒素對細胞的毒性原理以及細胞膜成份影響心臟毒素膜破壞活性的分子機制。我們利用微脂體並以螢光或顏色的變化觀察心臟毒素於脂質膜上作用的現象，發現脂質的飽和程度會影響心臟毒素的膜破壞活性以及膜結合構型，而飽和脂質組成的細胞膜不利於心臟毒素形成有效的膜破壞構型。同時發現心臟毒素除了以正負電荷與細胞膜結合外，亦以疏水性結合與脂質膜作用，而鞘磷脂能促進心臟毒素展現更多的膜破壞活性，並且心臟毒素的第二指環位置，在心臟毒素對含鞘磷脂的微脂體膜破壞活性的展現扮演重要角色。除了脂質外，O型血型醣基 (H抗原)亦會與心臟毒素3及心臟毒素5結合，而僅有心臟毒素3的膜破壞活性會受到H抗原抑制，與H抗原結合影響層面包括CTX3結構改變、抑制脂質膜結合量以及降低形成多聚體的能力，另一方面CTX5與H抗原結合雖然會些微改變CTX5結構，但不影響CTX5的膜破壞能力、脂質膜結合量以及形成多聚體的能力。除此之外發現脂質膜融合作用是心臟毒素造成細菌細胞膜成份微脂體膜破壞活性展現的原因，心磷脂會促進心臟毒素3的脂質膜融合能力，導致心臟毒素3對細胞膜成份含心磷脂之細菌的抗菌作用。總結上述結果，本研究對脂質飽和程度、鞘磷脂、血型醣基及細菌細胞膜組成，此四種細胞膜成份對心臟毒素的作用機制進行研究，進一步闡述心臟毒素膜破壞活性受其調控的現象，此結果有助於更全盤地瞭解心臟毒素在生物體組織扮演的角色功能並且可做為未來進行醫療應用的研究基礎。

Naja naja atra Cardiotoxins (CTXs), basic polypeptides of 60 amino acid residues adopt a three-fingered loop-folding topology and show cytotoxicity for human tissues in targeting cell membrane. Despite having highly similar sequence, the six CTX isoforms also display different cytotoxic potencies and hemolytic activities. The goal of these studies is to explore the mechanical processes that involved in membrane-damaging activities of CTXs on vesicles composed of different cell membrane components, and to delineate the events that lead to different biological activities of CTXs. The studies were performed by estimating the color transformation of phospholipid/polydiacetylene vesicles and the fluorescence enhancement of fluorescein-labeled phospholipid/protein or fluorescein released from vesicles. It was found that vesicles consisted of unsaturated phospholipids improve membrane-damaging activity of CTXs and adopt a vital membrane-bound conformation of CTXs. In contract, the characteristic of vesicles consisted of saturated phospholipids was against CTXs adopting an essential membrane-damaging structure. It was also found that not only electrostatic force but also hydrophobic force were involved in the interaction between CTXs and membrane. Comparing with phosphatidylcholine-only vesicles, CTXs displayed higher membrane-damaging activity for the sphingomyelin-containing vesicles, and the loop2 region of CTXs play a crucial role for the membrane-damaging activity of sphingomyelin-containing vesicles. Besides, the CTX3 and CTX5 would interact with the H-antigen of blood group O red blood cells, but only the binding of CTX3 with H-antigen reduce its membrane-damaging activity for red blood cells membrane. Moreover, the fusogenicity of CTXs is responsible for the membrane-damaging activity of CTXs toward bacterial membrane-mimicking vesicles. The cardiolipin have the potency to improve the fusogenicity of CTX3, which induced the bactericidal activity toward the cardiolipin-containing bacterium.

摘要 i
Abstract ii
目錄 iii
圖次 v
表次 viii
第一章 前言 1
心臟毒素蛋白 (cardiotoxin (CTX)) 1
微脂體 (liposomes) 3
圖及圖說 7
第二章 實驗材料與方法 11
I. 微脂體製備 11
II. 膜破壞活性試驗 12
III. 圓二色偏極光譜之結構分析 12
IV. CTXs形成多聚體試驗 13
V. 蛋白質結合單層大微脂體分析 (FPE) 13
VI. 蛋白質結合多層微脂體分析 14
VII. 脂質/聚聯炔 微脂體比色試驗 14
VIII. 溶血能力試驗 15
IX. 脂質膜流動性分析 16
X. 傅立葉式紅外線光譜分析 16
XI. 微脂體聚集試驗 17
XII. 脂質膜融合分析 17
XIII. 內層膜螢光能量轉移試驗 18
XIV. 酵素轉化血型醣基 18
XV. CTXs與醣基親和性分析 18
XVI. 化學修飾CTX的胺基酸殘基 19
第三章 研究目標 21
第一節 目標一 23
研究背景 23
結果與討論 25
圖及圖說 31
第二節 目標二 41
研究背景 41
結果與討論 43
圖及圖說 49
表格及說明 59
第三節 目標三 63
研究背景 63
結果與討論 65
圖及圖說 71
第四節 目標四 83
研究背景 83
結果與討論 87
圖及圖說 97
表格及說明 107
第四章 結論 109
參考文獻 113
附錄 131

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