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

Chitosan inhibits Candida albicans growth by decreasing secretory aspartyl proteinase activity

張高鳴(Ｋao-Ming Chang)

指導教授 ： 柯恩生

臺北醫學大學/口腔醫學院/牙醫學系碩博士班/碩士(2011年)

https://doi.org/10.6831/TMU.2011.00107

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。

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

並列摘要

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.

並列關鍵字

Candida albicans ； chitosan ； secretory aspartyl proteinase ； binding assay ； minimal inhibitory concentration ； growth curve ； western blotting ； Sap activity assay

參考文獻

14. 行政院農委會漁業署推廣月刊.242期:10-19.

1. Aida Pitarch RD-O, Gloria Molero, MercedePardo,Miguel Sánchez, Concha Gil, César Nombela. Analysis of the serologic response to systemic Candida albicans infection in a murine model. Proteomics 2001;1:550-59.

2. Kuriyama T, Williams DW, Lewis MA. In vitro secreted aspartyl proteinase activity of Candida albicans isolated from oral diseases and healthy oral cavities. Oral Microbiol Immunol 2003;18:405-7.

3. Naglik JR, Challacombe SJ, Hube B. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol Mol Biol Rev 2003;67:400-28, table of contents.

4. Schaller M, Borelli C, Korting HC, Hube B. Hydrolytic enzymes as virulence factors of Candida albicans. Mycoses 2005;48:365-77.