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研究生(外文):Ming-Li Chou
論文名稱(外文):Study on Morphological Changes of Candida albicans under Stress of Fluconazole and Terbinafine
外文關鍵詞:candida albicans
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白色念珠菌(Candida albicans)屬於不完全菌亞門(Deuteromycotina),為出芽型酵母,自然界中以二倍體(Diploid)的形式存在。直至今日,都尚未有關於白色念珠菌單倍體(haploid)及其減數分裂(meiosis)之相關報告,其基因之重組機制也只有擬有性世代(parasexual)的發現。由於白色念珠菌的mating locus與麵包酵母(Saccharomyces cerevisiae)具有相似的同源基因,因此大多都認為C. albicans較接近子囊菌。白色念珠菌在形態上自然會進行白色/渾濁之轉換(White/Opaque switching),其機率約為萬分之一,而此兩種形態相對之基因形則為異型合子(heterozygote, ex: a/α)及同型合子(homozygote, ex: a/a, α/α)。本實驗室曾發現Fluconazole及Terbinafine等抗真菌藥物會使得白色念珠菌白色/渾濁形態轉換之機率顯著提高至1.765x10-1,因此推測其中有減數分裂發生之可能性。本研究以光學顯微鏡(optical microscopy)觀察,經200 ug/mL Terbinafine處理之白色念珠菌株細胞形態,可於抑菌圈周圍發現到之多重出芽細胞群,為一個大的母細胞周圍帶有2至4個較小的子細胞(類似Snoopy掌印,尚稱Snoopy),而對照處理之細胞形態則為球狀細胞或單出芽型細胞,與一般細胞無異。研究進一步以共軛焦顯微鏡(confocal microscopy)觀察,經Terbinafine處理之白色念珠菌母細胞之細胞核變化,經由DAPI染色,在一到兩星期時大母細胞中有四核期的產生,且在三到四星期時可以看到母細胞呈現凹陷狀,無核,並將四個核送入新生之較小出芽子細胞中。此觀察結果與減數分裂之假設符合,可提供作為形態上之證據。此外本研究以掃描式電子顯微鏡觀察亦可以看到母細胞呈現凹陷狀的結果。同樣地,在穿透式電子顯微鏡下發現,母細胞與子細胞之間的出芽構造與控制組細胞不同。由以上型態之觀察結果傾向於C. albicans可能是擔子菌。因此可能推翻之前學者認為C. albicans接近子囊菌的假說。至於Azole及Allylamine等藥物會造成此一型態改變之機轉,則需進一步的探討。
Candida albicans is similar to Saccharomyces cerevisiae (Deuteromycotina), has a diploid genome and asexual life cycle in nature. Until now, there is no report to present sexual processing in C. albicans yet. Because of the fact that homologous genes of mating locus in C. albicans were identified to S. cerevisiae, previous studies revealed C. albicans belonged to Ascomycota. C. albicans processed a low frequency (under 10-4 ratio) white-opaque switch in nature, white phase is heterozygote and opaque phase is homozygote. The cloudy zone test results for multi-budding population of C. albicans via 200 ug/mL Terbinafine treated were observed by optical microscopy. 2-4 daughter cells surrounded a mother cell (snoopy’s paw-like), compared with the control group. Previous study in our lab (Ou et al., 2007), the phenomenon which C. albicans had a high frequency (above 1.765x10-1 ratio) white-opaque switch in anti-bacterial drugs treated condition such as Fluconazole or Terbinafine were observed, so, we presumed that meiosis may happened in C. albicans . Furniture study, nuclei change of C. albicans which were treated by Terbinafine via DAPI staining were observed by confocal microscopy.1-2 weeks, mother cells went through 4 nuclei phase and 3-4 weeks, mother cells presented a cuppy phenotype which had no nucleus, and the 4 nuclei transferred to the budding offspring. These data corresponded with our hypothesis, which provided a morphological evidence strongly. In addition, we could observe that mother cells presented cuppy phenotype by SEM technology in this study. Similarly, we could find out the peduncle structure between mother and daughter cells were different from the control group. According to these results, C. albicans might classify to basidium subkingdom. Accordingly, these findings might overthrow previous studies which are considered as the fact that C. albicans belong to Ascomycota. With respect to the changed type of mechanism of C. albicans via the effect of Azole or Allylamine, it need to be studied further more.
壹、前言 1
第一章 白色念珠菌 1
1-1 白色念珠菌的簡介 1
1-2 白色念珠菌的分類 2
1-3 研究動機 3
第二章 白色念珠菌的交配型態 4
2-1 白色念珠菌交配系統(mating system)的發現 4
2-2 白色念珠菌的表現形 4
2-3 白色念珠菌的交配 7
2-4 白色念珠菌交配與White-Opaque switching間的關係 8
2-5 白色念珠菌交配之細胞生物學 9
第三章 抗真菌藥物 11
3-1 抗真菌藥物種類和機制 11
3-2 白色念珠菌與Terbinafiner及Fluconazole 13
第四章 本研究相關之酵母菌 15
4-1 麵包酵母 15
4-2 新型隱球菌 15
第五章 本實驗室前期研究 16
第六章 論文研究動機與目標
6-1 研究動機 18
6-2 研究目的 19
貳、實驗架構 20
參、實驗材料與方法 21
一 材料 21
二 培養基 23
三 實驗方法 25
3-1抗真菌類藥物敏感性試驗對白色念珠菌opaque出現頻率之影響 25
3-2形態觀察 28
3-3 在光學顯微鏡下觀察多重出芽細胞與W/O表現形轉換頻率之相關性 34
肆、實驗結果 35
4-1 抗真菌類藥物敏感性試驗( E-test )對白色念珠菌opaque出現頻率之影響 35
4-2形態觀察 42
4-3 在光學顯微鏡下觀察多重出芽細胞與W/O表現形轉換頻率之相關性 58
伍、討論 62
陸、結論 72
柒、未來工作 73
捌、參考文獻 74
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66. 歐聰億等 台北醫學大學碩士論文 抗真菌藥Fluconazole對白色念珠菌臨床菌株形態學影響之探討
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