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研究生:林小琪
研究生(外文):Hsiao-Chi Lin
論文名稱:結合光動力殺菌與抗真菌藥物於白色念珠菌的生物效應探討
論文名稱(外文):Investigate the biological consequences of combining photodynamic inactivation and antifungal agents against Candida albicans
指導教授:陳進庭
口試委員:林晉玄吳亘承簡雄飛
口試日期:2017-06-20
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:50
中文關鍵詞:白色念珠菌光動力殺菌甲苯胺藍氟康唑5-氨基乙酰丙酸卡泊芬淨生物膜
外文關鍵詞:Candida albicansPhotodynamic inactivationToluidine blue OFluconazole5-Aminolevulinic acidCaspofunginBiofilm
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白色念珠菌(Candida albicans)為院內感染的伺機性真菌之一,目前治療真菌感染主要還是以抗真菌藥物為主,但大多有副作用及抗藥性問題產生。光動力殺菌Photodynamic inactivation (PDI)是一種用來解決菌體產生抗藥性問題的治療方式,其作用方式為藉由光感物質在光激發下產生單態氧及自由基來破壞菌體,進而導致菌體死亡。
實驗室先前針對懸浮培養的白色念珠菌以光動力結合抗真菌藥物Fluconazole,發現可提高Fluconazole的藥物作用,但在白色念珠菌生物膜以相同模式進行殺菌,卻無法提升殺菌效果。於此,本研究分別分析光動力殺菌後的生物膜菌體生長曲線、菌絲的生成能力、胞外聚合物厚度,以及配合caspofungin,推論光動力殺菌結合抗真菌藥物對白色念珠菌的生物膜殺菌效果不佳,可能和光動力殺菌的效果與抗真菌藥物的性質有關。另外以5-Aminolevulinic acid (5-ALA)進行光動力殺菌,結合抗真菌藥物也可在懸浮菌體提高殺菌效果。未來將以5-ALA來探討是否因粒線體受損導致菌絲生成受到影響,再加入抗真菌藥物可以提升殺菌效果,以探討光動力殺菌結合抗真菌藥物的殺菌機制。
Candida albicans is an opportunistic fungus in hospital infection. Antifungal drugs are mainly used for the treatment of fungal infection nowadays. However, there are side effects and drug resistance problems. Photodynamic inactivation (PDI) is one of the alternative treatments used to overcome the problem of drug resistance. The principle of PDI is to combine photosensitizer and light to produce reactive oxygen species that are toxic to pathogens.
Previous studies in our laboratory found that combination of PDI and Fluconazole could increase the antifungal activity against suspension of C. albicans; however, the effect is not significant against C. albicans biofilm. In this study, we analyzed the growth curves of biofilm, the ability of hyphae formation and the thickness of extracellular polymeric substances (EPS) after PDI. Meanwhile, another antifungal drug, caspofungin was used to combine with PDI to study the antifungal activity against suspension and biofilm of C. albicans. We found the ineffectiveness of PDI combined with fluconazole against biofilm might relate to the effect of PDI and the action mechanism of antifungal drugs. Furthermore, we found that the combination of a photosensitizer precursor, 5-Aminolevulinic acid (5-ALA), and antifungal drugs could also increase the antifungal effect against the suspension of C. albicans.
第一章 緒論 1
1.1 微生物與人類 1
1.1.1正常菌叢 1
1.1.2伺機性感染 1
1.1.3院內感染 2
1.2白色念珠菌 3
1.2.1白色念珠菌感染疾病 3
1.2.2白色念珠菌生物膜 4
1.2.3白色念珠菌生物膜生成過程 4
1.2.4白色念珠菌生物膜抗藥性機制 5
1.3抗真菌藥物 7
1.3.1抗真菌藥物種類 7
1.3.2抗真菌藥物的抗藥性 8
1.4光動力治療 11
1.4.1光動力治療發展與歷史 11
1.4.2光動力治療作用機轉 11
1.4.3光感物質 12
1.4.4光動力殺菌 13
1.5研究動機與目的 14
1.6研究架構 15
第二章 材料與方法 16
2. 1藥品 16
2.2儀器 16
2.3菌種來源、保存與活化 17
2.3.1菌種來源 17
2.3.2菌種保存 17
2.3.3菌種活化 17
2.4實驗方法 18
2.4.1藥物配置 18
2.4.2白色念珠菌懸浮菌體培養 18
2.4.3白色念珠菌生物膜培養 18
2.4.4光動力殺菌於白色念珠菌生物膜 18
2.4.5白色念珠菌生物膜生長曲線 18
2.4.6光動力殺菌於白色念珠菌懸浮菌體 19
2.4.7誘導白色念珠菌菌絲生成分析 19
2.4.8生物膜胞外聚合物觀察 19
2.4.9光動力殺菌結合抗真菌藥物於白色念珠菌生物膜 19
2.4.10以5-ALA進行光動力殺菌結合抗真菌藥物於白色念珠菌懸浮
菌體 19
2.4.11統計分析 20
第三章 結果 21
3.1光動力殺菌後白色念珠菌生物膜之生長曲線 21
3.2光動力殺菌後白色念珠菌菌絲生成能力分析 21
3.3光動力殺菌後白色念珠菌生物膜胞外聚合物厚度分析 22
3.4光動力殺菌結合caspofungin對白色念珠菌生物膜影響 22
3.5光動力殺菌結合posaconazole對白色念珠菌生物膜影響 23
3.6以不同濃度5-ALA進行光動力殺菌之效果 23
3.7 5-ALA進行光動力殺菌結合不同濃度抗真菌藥物之效果 24
第四章 討論 25
4.1光動力殺菌後白色念珠菌生物膜之生長曲線探討 25
4.2光動力殺菌後白色念珠菌菌絲生成能力分析 25
4.3光動力殺菌後白色念珠菌生物膜胞外聚合物厚度分析 26
4.4光動力殺菌結合caspofungin對白色念珠菌生物膜效果探討 27
4.5光動力殺菌結合posaconazole對白色念珠菌生物膜效果探討 28
4.6 5-ALA進行光動力殺菌結合不同濃度抗真菌藥物之效果探討 28
4.7生物膜狀態之探討 29
第五章 結論與未來研究方向 31
結果圖表 32
附錄 41
參考文獻 43
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