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研究生:謝尚豪
研究生(外文):Shang-hao Hsieh
論文名稱:Indolicidin 及其類似物與微脂粒交互作用之焓測量
論文名稱(外文):Interaction of Indolicidin and its analogues with Model Membrane-enthalpy measurement
指導教授:阮若屈
指導教授(外文):R.C. Ruaan
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:72
中文關鍵詞:抗菌胜肽膜擾亂恆溫滴定微卡計
外文關鍵詞:IndolicidinFluorescence AnisotropyIsothermal titration calorimeter
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Indolicidin為一有潛力的抗生藥物,其具有相當廣泛的抗生活性,對抗細菌、真菌、病毒皆有不錯的能力,且是一段具有不錯經濟效益的短鏈胜肽。然而,Indolicidin對人類紅血球的溶血活性卻限制其進一步的發展。雖然,擁有低溶血血性的IL之類似物(ILK7、ILF89、ILK7F89)已經被設計,但它們的行為機制仍然不是非常清楚。本研究主要是探討其生物活性與膜擾亂之間的關係。我們藉由螢光異位向性實驗研究膜擾亂的情形,並以恆溫滴定微卡計研究胜肽與膜之間的熱變化。而我們使用兩種仿生物細胞膜的微脂粒,仿細菌細胞膜(POPG/POPC=1, PG/PC-SUVs)與仿人類紅血球細胞膜(純 POPC, PC-SUVs)。而我們進一步探討IL及其類似物對抗革蘭氏陽性菌(staphylococcus epidermidis)的活性,結果發現IL的類似物皆擁有較IL更強或相當的抗菌活性。另一方面,在POPC微脂粒的作用中,胜肽對膜的擾亂與膜擾亂焓和其溶血活性沒有很強烈的關係,所以對於溶血活性主要仍是與其胜肽吸附量有關。因此,對於抗菌活性胜膜的擾亂程度確實是一很重要的影響因素。
Indolicidin (IL) is a cationic antibacterial peptide with broad-spectrum against many pathogens; therefore, it is a potential peptide antibiotic. However, the hemolytic activity of IL limits its clinical application. Although the IL-analogues (ILK7, ILF89 and ILK7F89) with lower hemolysis have been designed, the action mechanisms of them were still under debating. In this study, we tried to examine the relationships between bioactivity and membrane perturbation. The membrane perturbation was investigated through the help of fluorescence anisotropic measurement. The membrane association enthalpy and membrane perturbation enthalpy were examined by isothermal titration calorimeter (ITC) and peptide-membrane adsorption isotherm. Two types of small unilamellar vesicles (SUVs) were used to mimic the bacterial-like membrane (POPG/POPC=1) and erythrocyte-like membrane (pure POPC). The antibacterial activity of IL and its analogues against Gram-positive bacteria (staphylococcus epidermidis) were also examined. The results revealed that IL-analogues with enhancing or similar antibacterial activity than that of IL peptide.
The interaction of peptide and PG/PC-SUVs revealed that the peptide with high antibacterial activity owns strongly membrane perturbation and highly membrane perturbation enthalpy. On the contrary, as peptide and PC-SUVs interaction, the membrane perturbation of peptide action and membrane perturbation enthalpy are not strongly correlated with its hemolytic activity. Particularly, the hemolysis is related to the amounts of peptide adsorption. Consequently, the membrane perturbation suffered by peptide is an important implication on antibacterial activity.
中文摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 viii
第一章 緒論 1
1-1 研究動機 1
1-2 研究目的 2
二. 文獻回顧 3
2-1 鹼性抗生胜肽 3
2-1-1 鹼性抗生胜肽的抗生機制 4
2-2 鹼性抗菌胜肽Indolicidin 7
2-2-1微脂粒 7
2-2-2 Indolicidin 抗菌活性及機制 9
2-2-3 Indolicidin 的溶血活性及機制 11
2-3 Indolicidin的類似物 11
2-4 胜肽在細胞膜上之行為與對膜之影響的探討 13
2-4-1 利用圓二色光譜探討胜肽與膜作用之結構變化 13
2-4-2 利用螢光光譜探討胜肽在膜上之分散程度 15
2-4-3 用螢光光譜探討胜肽在膜上之插入深度 15
2-4-4 探討抗生胜肽對細胞膜的擾亂程度 15
2-5恆溫滴定微卡計(Isothermal titration calorimeter)簡介與原理 17
2-4-1胜肽與微脂粒交互作用之焓值測量 18
第三章 實驗藥品、設備與方法 20
3-1 實驗藥品 20
3-2 實驗設備 21
3-3 微脂粒之製備 22
3-4 抗菌活性 23
3-4-1 單一菌落的培養 23
3-4-2 菌液預培養 24
3-4-3抗菌活性 24
3-5恆溫滴定微卡計實驗(MicroCal VP-ITC) 24
3-6 IL及其類似物吸附曲線最適化-得其吸附量 27
3-7含有 DPH之POPC 微脂粒的製備 29
3-8 Indolicidin 及其類似物螢光異位相性 30
第四章結果與討論 31
4-1 Indolicidin 及其類似物的抗生活性 31
4-1-1抗S. epidermidis活性 32
4-1-2 Indolicidin及其類似物抗生活性討論 33
4-2 Indolicidin 及其類似物POPG/POPC微脂粒交互作用 36
4-2-1 以螢光異位向法探討IL及其類似物對POPG/POPC膜擾亂程度 36
4-2-2 IL及其類似物對POPG/POPC微脂粒交互作用之焓測量 38
4-2-2-1 IL及其類似物對POPG/POPC微脂粒之吸附量 40
4-2-2-2 IL及其類似物對POPG/POPC微脂粒之累積熱 41
4-3 Indolicidin 及其類似物POPC微脂粒交互作用 45
4-3-1 以螢光異位向法探討IL及其類似物對POPC膜擾亂程度 45
4-3-2 IL及其類似物對POPC微脂粒交互作用之焓測量 47
4-3-2-1 IL及其類似物對POPC微脂粒之吸附量 49
4-3-2-2 IL及其類似物對POPC微脂粒之累積熱 50
第五章 結果與討論 53
第六章 參考文獻 54
第七章 附錄 57
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