臺灣博碩士論文加值系統

白色念珠菌為具有雙形性的真菌，是人類主要的伺機性病原真菌之一，可引起黏膜及深層組織的感染。近年來研究指出白色念珠菌可以在許多植入性人工醫材上形成生物膜，而由於生物膜的構造與特性，使得抗藥性增加且有利於躲避宿主的免疫系統攻擊，進而導致高死亡率，因此尋求有效預防或對抗生物膜的新方法，做為替代抗生素或減少抗生素的使用量，是急待努力的方向之一。精油又稱為揮發油，是從植物萃取而來的油狀芳香液體，富含萜類化合物。許多精油已被證實具有抗細菌及真菌的能力，但其對於抗真菌生物膜的研究仍不多。本研究的目的，就是希望從植物精油中，篩選出可以抑制白色念珠菌生物膜的活性物質並探討其所影響之分子層次。篩選結果發現，花椒精油及其主要成分linalool具有抑制白色念珠菌生物膜形成及清除已生成24小時之白色念珠菌生物膜的能力；此外，linalool具有抑制白色念珠菌形成發芽管的能力。在分子作用機制方面，我們發現linalool可降低adhesin基因表現量，包含HWP1及ALS3；菌絲形成的主要調控路徑cAMP-PKA pathway中的CYR1及MAPK pathway中的CPH1，以及其共同上游RAS1之基因表現量均受到linalool的抑制。而與hyphae長時間維持相關的基因：UME6、HGC1及EED1，其表現量亦會受到linalool抑制。由本研究的結果，linalool具有做為抑制白色念珠菌生物膜所引起的感染的應用價值。

Biofilms are structured microbial communities in which the cells bind tightly to a surface and become embedded in a matrix of extracellular polymeric substances produced by these cells, and characteristically display a phenotype that is markedly different from that of planktonic cells. Candida albicans, the dimorphic yeast, is a major human fungal pathogen that causes both mucosal and deep tissue infections. Biofilm-associated Candida albicans infections are clinically relevant due to their high levels of resistance to traditional antifungal agents. Essential oils are aromatic oily liquids obtained from plant materials that are mainly composed of terpenes and some other non-terpene components. The essential oils of many aromatic plants are known for their antifungal properties, but their antibiofilm activity has not been studied extensively. The aim of this study is to investigate whether essential oils and its components could inhibit the development of C. albicans biofilms. In our screen system, biofilms were formed on microtiter plates and quantified by XTT reduction assay. After preliminary screening, several candidate essential oils and their major components were found to inhibit C. albicans biofilm formation and linalool was chosen for further study. Linalool exhibited antifungal activity against C. albicans ATCC 14053, with a minimum inhibitory concentration of 8 mM. Sub-MIC concentrations of linalool also inhibited the formation of germ tubes and biofilms in that strain. The defective architecture of C. albicans biofilms exposed to linalool was characterized by scanning electron microscopy. The expression levels of the adhesin genes HWP1 and ALS3 were downregulated by linalool, as assessed by real-time RT-PCR. The expression levels of CYR1 and CPH1, which encode components of the cAMP-PKA and MAPK hyphal formation regulatory pathways respectively, were also suppressed by linalool, as was the gene encoding their upstream regulator Ras1. The expression levels of long-term hyphae maintenance associated genes, including UME6, HGC1, and EED1, were all suppressed by linalool. These results indicate that linalool may have therapeutic potential in the treatment of candidiasis associated with medical devices because this compound interferes with the morphological switch and biofilm formation of C. albicans.

中文摘要 1
Abstract 2
第一章、緒論 4
一、白色念珠菌 4
二、C. albicans的形態調控 6
三、群感效應與形態轉換 8
四、生物膜 11
五、C. albicans生物膜 13
六、抗真菌劑 16
七、抗生物膜的方法 18
八、植物精油 19
九、研究動機與目的 21
十、實驗架構與策略 23
第二章、材料與方法 24
一、實驗材料 24
1. 菌株 24
2. 培養基 24
3. 精油 24
4. 化學藥品與耗材 24
5. 儀器設備 25
二、實驗方法 26
1. Candida spp.之保存與培養 26
2. C. albicans生物膜之培養 26
2. C. albicans生物膜之偵測 27
3. 氣相層析質譜儀分析 27
4. 最低抑菌濃度及最低殺菌濃度測定 28
5. 殺菌時間試驗 28
6. 發芽管觀察 28
7. RNA萃取與即時定量PCR (quantitative real time-PCR) 29
8. 掃瞄式電子顯微鏡觀察 29
第三章、結果 31
一、抑制C. albicans生物膜形成之植物精油篩選結果 31
二、Candida spp. 對於linalool的感受性 31
三、殺菌時間曲線 32
四、Linalool對於C. albicans發芽管形成的影響 33
五、Linalool對C. albicans生物膜之影響 33
六、Linalool對於C. albicans生物膜相關基因表現之影響 34
第四章、討論 36
一、Linalool具有對抗C. albicans生物膜之潛力 36
二、Linalool具有抑制C. albicans菌絲形成以及長期維持的作用 37
三、成熟的C. albicans生物膜對於linalool仍具有感受性 38
四、Linalool抑制C. albicans的黏附能力 38
五、Linalool抑制C. albicans生物膜的作用機轉 39
六、Linalool的安全性探討 40
七、總結 41
第五章、參考文獻 42
第六章、圖表 57
表一、Real time-PCR 引子序列 57
表二、精油名稱與主要成份列表 58
表三、C. albicans對於精油成分的感受性 59
表四、Candida spp.對linalool及amphotericin B的感受性 60
圖一、精油對於C. albicans生物膜之影響 61
圖二、蓽澄茄精油GC/MS分析圖譜 62
圖三、玫瑰草精油GC/MS分析結果 63
圖四、花椒精油GC/MS分析結果 64
圖五、香茅精油GC/MS分析結果 65
圖六、香薷精油GC/MS分析結果 66
圖七、檸檬香茅精油GC/MS分析結果 67
圖八、化合物結構圖 68
圖九、精油成份對於C. albicans生物膜之影響 69
圖十、Linalool及amphotericin B對C. albicans的殺菌時間曲線 70
圖十一、Linalool抑制C. albicans發芽管的形成 71
圖十二、Linalool對於C. albicans生物膜之影響 72
圖十三、以掃描式電子顯微鏡觀察C. albicans生物膜 73
圖十四、Linalool對於C. albicans生物膜基因表現的影響 74
附圖一、調控C. albicans形態轉換的訊息傳遞途徑 75
附圖二、C. albicans菌絲誘導與維持之分子調控 76
附圖三、C. albicans生物膜發展過程示意圖 77

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