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研究生： 林達翰
Da-han Lin
論文名稱： Indolicidin及其類似物的聚集行為及其與仿生細胞膜間之交互作用
Oligomerization behavior of Indolicidin and its analogues and its interaction with bio-mimic membranes
指導教授： 阮若屈
Ruoh-chyu Ruaan
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度： 99
語文別： 中文
論文頁數： 119
中文關鍵詞： 磷脂微脂粒滲漏 、仿生細胞膜 、分子動態模擬 、凝膠電泳 、胜肽寡聚 、鹼性抗生胜肽
外文關鍵詞： Indolicidin, antimicrobial peptide, electrophoresis, molecular dynamics simulations, dye leakage, peptide oligomerization
相關次數： 點閱：13  下載：0 
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    • 鹼性抗生胜肽Indolicidin (IL) 因具有廣泛且迅速之抗菌活性，甚至可以對抗病毒與癌細胞，也不易造成微生物抗藥性，因此被認為是極具有潛力的抗生藥物。然而，Indolicidin對人體紅血球的溶血活性，限制了其在臨床應用上之發展。本實驗室曾利用分子動態模擬設計了一連串的Indolicidin類似物：IL-K7、IL-F89、IL-K7F89，設計出具有高抗菌、低溶血之抗生胜肽，但對於此類胜肽何以導致溶血又何以能抗菌並不能完全了解。先期的研究發現，Indolicidin及其類似物在水溶液中似乎有寡聚現象，而其寡聚程度與其溶血活性正相關，因此本論文之研究目的在於探討Indolicidin及其類似物的寡聚現象以及寡聚現象對於仿生物細胞膜的影響。
    我們利用凝膠電泳與分子模擬探討Indolicidin及其類似物的寡聚行為，結果發現Indolicidin及其類似物最多由三條胜肽形成聚集。然後我們測試Indolicidin及其類似物與仿生微脂粒間的作用。我們使用兩種微脂粒，一種完全由POPC組成，表面呈現電中性；另一種由POPC與POPG混合物形成，表面帶負電荷。結果發現這些胜肽對兩種仿生物細胞微脂粒作用情形類似，皆能使其產生破洞，破洞程度也隨著濃度的增加而增加。然而當我們使用文獻中所載低溶血性的SAP胜肽時，則發現SAP並不會造成帶負電荷的POPC/POPG微脂粒的破壞，由此可說明SAP的抗菌性不是經由破壞細胞膜而產生。我們又以分子模擬觀察Indolicidin寡聚體在兩種仿生細胞膜上不同位置造成膜雙層擾動的情形。由結果發現，Indolicidin寡聚體確實會會對磷脂雙層膜造成擾動，甚至在膜中心有些微水分子滲入，說明可能有短暫孔洞的產生。
    由以上研究，我們猜測Indolicidin及其類似物所形成之寡聚物，在吸附於紅血球細胞膜後，會造成短暫的孔洞，而此短暫孔洞的形成可能與其溶血活性有關。

    Cationic antimicrobial peptide –Indolicidin (IL) has been considered to be a potential antibiotic drug by its broad-spectrum of antibiotic activity against bacteria, fungi and even viruses. However, the hemolytic activity limits its clinical applications. To reduce its hemolytic activity, we had designed several less hemolytic Indolicidin analogues, IL-K7, IL-F89, IL-K7F89, through MD simulations in our previous work. But the mechanisms of its antibiotic and hemolytic behaviors are stIndolicidinl not clear. We had found that these peptides might oligomerized in aqueous solution and the degree of peptide oligomerization was consisted with their hemolytic activity. Therefore, we try to first study the possible structure of peptide oligomerization. And then the interactions between peptide oligomers and bio-mimic membranes are under investigation.
    We studied the size of oligomer by gel electrophoresis and molecular dynamic simulation. The results showed that Indolicidin and its analogues might form dimer or trimer. We then studied the interaction between peptides and bio-mimic membranes by calcium dye leakage experiment. Two types of bio-mimic liposomes were made. One was made of pure POPC lipid of which the surface was neutral, the other was made of the mixture of POPC and POPG of which the surface was negatively charged. We found that both types of liposomes were perturbed by the peptides and the degree of dye release was increased with the amount of peptide added. Surprisingly, no dye leakage was observed when we test the peptide SAP, a well-known antimicrobial peptide of low hemolytic activity. The result indicated that the antimicrobial activity of SAP was not caused by cell membrane perturbation.
    We further investigate the interaction between Indolicidin trimmers and bio-mimic membranes by all-atom molecular dynamic simulation. It was found that the Indolicidin trimmer dissociated and then re-associated in the POPC/POPG membrane. Simultaneously, we found that water molecules entered the hydrophobic core of membrane. The result supported the possibility of transient pore formation.
    All the results indicated that Indolicidin and its derivatives might form dimmers or trimmers in the aqueous solution. The oligomers adsorbed onto the membrane and perturb the membrane structure. We considered that the membrane perturbation by peptide oligomers was related to the hemolytic behavior of these peptides.

    摘要 i Abstract iii 第一章 緒論 1 1.1 研究動機 1 1.2 研究目的 2 第二章 文獻回顧 3 2.1 鹼性抗生胜肽 3 2.1.1 鹼性抗生胜肽簡介 3 2.1.2 鹼性抗生胜肽的發展及研究 3 2.1.3 鹼性抗生胜肽的抗生機制 4 2.2 鹼性抗生胜肽Indolicidin 9 2.2.1 Indolicidin的研究與發展 9 2.2.2 Indolicidin的生物活性 10 2.2.2.1 Indolicidin的抗菌活性及機制 10 2.2.2.2 Indolicidin的溶血活性及機制 14 2.3 Indolicidin與仿生物細胞膜之交互作用 15 2.3.1 細胞膜 15 2.3.1.1 細胞膜的基本性質 15 2.3.1.2 細胞膜的組成與結構 16 2.3.2 微脂粒 19 2.3.2.1 微脂粒的結構 19 2.3.2.2 微脂粒的分類 20 2.3.2.3 以微脂粒做為仿生物細胞膜 21 2.3.3 Indolicidin與微脂粒作用之相關研究 22 2.3.3.1 Indolicidin中Tryptophan的自身螢光光譜 22 2.3.3.2 螢光淬滅探討Indolicidin在膜內的深度 22 2.3.3.3 利用 Dye leakage探討Indolicidin對磷脂質膜的擾動 23 2.4 影響Indolicidin生物活性之因素 25 2.4.1 電荷 25 2.4.2 二級結構 25 2.4.3 疏水性 26 2.4.4 聚集行為 27 2.4.4.1 大小排斥層析 (Size exclusion chromatography) 27 2.4.4.2 毛細電泳 (Capillary electrophoresis) 28 2.4.4.3 螢光光譜 28 2.4.4.4 光散射 (Light scattering) 28 2.4.5 以凝膠電泳觀測蛋白質聚集行為 29 2.4.6 Indolicidin及其類似物之設計 31 2.4.6.1 Indolicidin類似物的設計 31 2.4.6.2 Indolicidin及其類似物的生物活性 32 2.5 分子動態模擬 (Molecular Dynamics simulation) 35 2.5.1 概述 35 2.5.2 分子動態模擬原理 35 2.5.3 分子動態模擬的計算程序 38 2.5.3.1 以牛頓力學計算作用力 38 2.5.3.2 Verlet 積分法 39 2.5.4 CAPs與磷脂膜作用之分子動態模擬 40 第三章 實驗藥品、儀器設備及方法 42 3.1 實驗藥品 42 3.2 實驗儀器 45 3.3 實驗方法 46 3.3.1 實驗架構 46 3.3.2 凝膠電泳 47 3.3.2.1 胜肽樣品製備 47 3.3.2.2 電泳膠片與相關溶液製備 48 3.3.3 SAP的生物活性 54 3.3.3.1 SAP溶血活性 54 3.3.3.2 SAP抗菌活性 54 3.3.4 Calcein 染劑滲漏實驗 57 3.3.4.1 SUVs、Calcein-SUVs製備與純化 57 3.3.4.2 Indolicidin及其類似物與Calcein微脂粒之交互作用 60 3.3.4.3 Calcein滲漏程度計算 61 3.3.5 分子動態模擬 62 3.3.5.1 系統的建立與參數設定 62 3.3.5.2 模擬結果分析 65 第四章 結果與討論 67 4.1 Indolicidin在水相系統中聚集行為探討 67 4.1.1 凝膠電泳 67 4.1.2 分子動態模擬結果 68 4.2 生物活性與Calcein螢光滲漏實驗 71 4.2.1 溶血活性 (人類紅血球細胞) 71 4.2.2 POPC微脂粒之Calcein螢光滲漏結果 71 4.2.3 抗菌活性 (E. coli) 72 4.2.4 POPC/POPG微脂粒之Calcein螢光滲漏結果 73 4.3 Indolicidin三聚體在膜上分散行為探討 79 4.3.1 Indolicidin三聚體在POPC膜上的行為 79 4.3.2 Indolicidin三聚體在POPC/POPG膜上的行為 83 4.3.3 POPC/POPG雙層膜頭基擾亂情形 83 4.3.4 POPC/POPG雙層膜中水分子分佈 84 第五章 結論 92 第六章 參考文獻 94

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