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系統識別號 U0007-2501201112184900
論文名稱(中文) 幾丁聚醣藉由抑制分泌性天冬氨酸蛋白酶活性來抑制 白色念珠菌之生長
論文名稱(英文) Chitosan inhibits Candida albicans growth by decreasing secretory aspartyl proteinase activity
校院名稱 臺北醫學大學
系所名稱(中) 牙醫學系碩博士班
系所名稱(英) School of Dentistry
學年度 99
學期 1
出版年 100
研究生(中文) 張高鳴
研究生(英文) Kao-Ming Chang
學號 M204097006
學位類別 碩士
語文別 中文
口試日期 2011-01-20
論文頁數 50頁
口試委員 指導教授-柯恩生
委員-王正怡
委員-蘇慶華
中文關鍵字 白色念珠菌  幾丁聚醣  分泌性天冬氨酸蛋白酶  結合試驗  最低抑菌濃度  生長曲線  西方墨點法  活性測試 
英文關鍵字 Candida albicans  chitosan  secretory aspartyl proteinase  binding assay  minimal inhibitory concentration  growth curve  western blotting  Sap activity assay 
學科別分類
中文摘要 白色念珠菌是人體內主要的真菌病原,尤其在免疫抑制的病人身上常見,現今治療白色念珠菌感染的藥物都有一些缺點,如毒性、副作用等;幾丁聚醣做為一個天然的化合物,擁有良好的生物相容性、低副作用以及產量豐富等特性,是一個具有潛力的抗真菌物質。本實驗的目的是測試幾丁聚醣抑制白色念珠菌的生長是否和Sap(secretory aspartyl proteinase)有關。首先我們使用抗菌實驗(MIC)確認幾丁聚醣抗菌效果,再來使用細胞存活性試驗觀察幾丁聚醣直接殺菌的可能性;接著使用西方墨點法來確認白色念珠菌培養在不同氮素來源之培養基中,是否會產生Sap;並且在生長曲線實驗中,測試幾丁聚醣對白色念珠菌生長的影響,同時也觀察幾丁聚醣對測試培養液的上清液中Sap活性的影響;最後以結合試驗(binding assay)測試幾丁聚醣是否會和Sap產生某種結合關係。
因此我們推測在適當的濃度下,幾丁聚醣可以藉由和Sap結合而降低其活性進而抑制白色念珠菌的生長。但它們之間的關係,仍需要更多的實驗的來證明之。
英文摘要 Candida albicans is the most common clinical fungal pathogen , especially in immunocompromised patients. There are some disadvantages of current antifungal drugs, such as toxicity, side-effect. Chitosan, a natural polymer with high biocompatibility, low side-effect and abundant source characters, may act as a potential antifungal substance. The purpose of this study was to determine whether chitosan inhibits Candida albicans growth and its possibility that secretory aspartyl proteinase(Sap) was involved. First, based on guidelines of NCCLS M27-A2, MIC was performed to test antifungal effect of chitosan, followed by cell viability test to verify fungicidal ability of chitosan. Western blotting was performed to test the Sap production of Candia albicans growing in different culture media. Inhibition of Candida albicans growth by chitosan was tested by growth curve. Sap activity assay was performed to detect the chitosan effect on Sap activity.
Finally, binding ability of chitosan to Sap was verified by binding assay.
These results suggested at proper concentration, chitosan inhibited Candida albicans growth by binding to Sap and leading to the decrease of its activity. Precise mechanism needs more future study.
論文目次 目 錄

頁 數
致 謝 .......................................... I
中文摘要 .......................................... III
英文摘要 ..........................................  V
目 錄 .......................................... VII
表 目 錄 .......................................... X
圖 目 錄 .......................................... XI
第一章 緒論
  第一節 研究動機 .............................. 1
第二節 研究目的 .............................. 8
第三節 研究假說 .............................. 8

第二章 文獻查證 .................................. 9

第三章 研究材料與方法
第一節 材料 .................................. 12
第二節 菌種與培養條件 ........................ 15
第三節 培養基,膠與溶液製備 ..................... 15
第四節 研究儀器 .................................. 19
第五節 研究架構 .................................. 20
第六節 最低抑菌濃度 .......................... 21
第七節 細胞存活性試驗 ........................ 21
第八節 Sap 活性試驗 .......................... 22
第九節 幾丁聚醣對白色念珠菌生長之影響 ........ 23
第十節 幾丁聚醣對Sap活性之影響 ............... 23
第十一節 結合試驗 ............................. 24
第十二節 統計方法 ............................. 25

第四章 分析與結果
第一節 最低抑菌濃度 .......................... 26
第二節 細胞存活性試驗 ........................ 26
第三節 Sap活性試驗 ........................... 27
第四節 幾丁聚醣對白色念珠菌生長之影響 ........ 28
第五節 幾丁聚醣對Sap活性之影響 ............... 29
第六節 結合試驗 .............................. 30

第五章 討論 ...................................... 31

第六章 結論 ...................................... 35

參考資料 .......................................... 36

附錄 .............................................. 39


表 目 錄

表一 SAP基因在白色念珠菌致病過程中所扮演的角色 .... 39
表二 幾丁聚醣對真菌之最低抑菌濃度 ................. 40
表三 幾丁聚醣對細菌之最低抑菌濃度 ................. 41
表四 幾丁聚醣的應用 ............................... 42

圖 目 錄

圖一 幾丁質與幾丁聚醣單體之結構 .................... 43
圖二 最低抑菌濃度試驗 .............................. 44
圖三 細胞存活性試驗 ................................ 45
圖四 Sap分泌試驗 ................................... 46
圖五 幾丁聚醣對生長在YCB-BSA培養基中的白色念珠菌之
影響........................................... 47
圖六 幾丁聚醣對生長在YCB-peptone培養基中的白色念珠菌
之影響 ........................................ 48
圖七 幾丁聚醣對Sap活性之影響 ....................... 49
圖八 結合試驗 ...................................... 50
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