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研究生: 王立蓁
Wang, Li-Chen
論文名稱: 研究膽固醇與醣化膽固醇影響細胞膜之流動性
Investigation of cholesterol and cholesteryl glucoside in influencing membrane fluidity
指導教授: 王雯靜
Wang, Wen-Ching
口試委員: 王雯靜
林立元
許宗雄
彭慧玲
張晃猷
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 分子與細胞生物研究所
Institute of Molecular and Cellular Biology
論文出版年: 2011
畢業學年度: 99
語文別: 中文
論文頁數: 53
中文關鍵詞: 薑黃素細胞膜流動性
相關次數: 點閱:4下載:0
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  • Helicobacter pylori is a Gram-negative microaerophilic gastric pathogen that infects approximately one-half of the human population. Infection withH. pylori is associated with gastritis, ulcerations, and gastric cancer.The common therapy for H. pylori infections is triple therapy that includes two kinds of antibiotics and a proton pump inhibitor. Given the high use of the antibiotic treatment, there is a decreasing rate in eliminating the infection in most countries.Curcumin is a natural yellow coloring polyphenol agent present in the spice turmeric (Curcuma longa), exhibits anti-inflammatory, antineoplastic, anti-oxidant and chemopreventive activities and has been demonstrated the effects of curcumin on the serum cholesterol and lipid peroxide levels. The membrane of biological cells or intra-cellular organelles plays an important role in maintaining cellular physiological functions. Here we utilized curcumin to treat AGS cells to evaluate the change in the membrane fluidity and to evaluate whether internalized of H. pylori was influenced (H. pylori internalization) by curcumin treatment. Results from amoxicillin protection assay showed that there was reduced colony forming numbers of bacteria when cells were treated with curcumin. We also observed a higher level of amoxicillin in the cytosol for curcumin-treated cells. Finally we applied fluorescence correlation spectroscopy (FCS) that is a technique to study molecular dynamics and mobility at single molecule level in solution. Our results revealed that curcumin treated cells exhibited a higher diffusion coefficient than did cells without treatment. Our results together suggested that curcumin influenced the membrane fluidity, thereby bacterial internalization.
    We have also analyzed the effect of cholesteryl-α-D-glucopyranosides (αCGs) by cholesterol-α-glucosyltransferase on the membrane fluidity. We have prepared a fluorophore-labeld αCG –incorporated liposomes. Fluorescence correlation spectroscopy analysis revealed that the addition of αCGs into the liposome led to an increased value of diffusion coefficient of DiIC18. Our results revealed that αCGs-treated liposome or cells exhibited a higher diffusion coefficient than did cells without treatment. Our results together suggested that αCG influenced the membrane fluidity.


    摘要 I Abstract III 誌謝 V 第一章 文獻回顧 1 1.1. 胃幽門螺旋菌 1 1.1.1. 胃幽門螺旋菌的發現歷史 1 1.1.2. 胃幽門螺旋菌的形態學特徵 1 1.1.3. 胃幽門螺旋菌之基因體 2 1.1.4. 胃幽門螺旋菌之流行病學 2 1.1.5. 胃幽門螺旋菌之感染檢測與治療方法 3 1.2. 細胞膜流動性 4 1.3. 測量膜流動性之方法介紹 5 1.4. 螢光相關光譜 7 1.4.1. 發展歷史 7 1.4.2. 原理介紹 9 1.5. 脂質體 10 1.6. 薑黃素 11 1.6.1. 薑黃素的發現及特性 11 1.6.2. 薑黃素對於膽固醇之生合成的影響 11 1.7. 醣化膽固醇 12 1.8. 研究動機與目標 13 第二章 實驗材料及方法 14 2.1. 胃幽門螺旋菌之培養 14 2.2. 人類胃腺癌細胞之培養 14 2.3. 螢光含醣膽固醇之萃取 14 2.3.1. 螢光含醣膽固醇之分離 14 2.3.2. 利用薄層分析法純化螢光醣化膽固醇 15 2.3.3. 醣化膽固醇之相對定量 15 2.4. Amoxicillin protection assay. 16 2.5. 螢光相關光譜 16 2.5.1. 螢光相關光譜系統之架設 16 2.5.2. 系統校正方法 17 2.6. 脂質體的製備 18 2.7. 脂質體的螢光相關光譜之偵測 19 2.8. 人類胃腺癌細胞的螢光染色製備 20 2.9. 人類胃腺癌細胞的膜流動性偵測 20 2.10. 藥物毒性測試 21 2.11. 膽固醇含量變化測試 21 2.11.1. 膽固醇濃度測驗試劑之作用原理 22 2.12. 免疫螢光染色法 22 第三章 結果 23 3.1. 薑黃素對細胞不具毒性 23 3.2. 薑黃素處理之後會降低細胞膽固醇的含量 23 3.3. 薑黃素處理之後可以提高抗生素殺菌的效果 24 3.4. 薑黃素處理之後可增加抗生素進入細胞的含量 25 3.5. 薑黃素處理之後提可以高細胞膜的流動性 26 3.6. 不同濃度的膽固醇及醣化膽固醇的膜流動性變化 26 3.7. 醣化膽固醇處理之後可以提高細胞膜的流動性 27 第四章 討論 28 4.1. 薑黃素劑量選擇與細胞毒殺性 28 4.2. 脂質體的製備與觀察 28 4.3. 細胞膜流動性的測量方法之選擇 29 4.4. 螢光相關光譜的測定 29 4.5. 醣化膽固醇對於細胞膜流動性的影響 30 參考文獻 32 表格與附圖 37

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