臺灣博碩士論文加值系統

微生物於自然界主要以生物膜 (biofilms) 方式存在,但生物膜之抗藥性機制使得一般抗生素療法成果不佳,因此開發新型生物膜防治法為當務之急。光動力療法 (photodynamic therapy, PDT) 為利用特定波長光源將光感物質 (photosensitizer) 激發至高能激發態,藉電子或能量轉移而產生自由基或單態氧 (1O2) 以傷害菌體,目前已應用於癌症或微生物防制。早期光動力抑菌實驗多著重於懸浮菌體,近年來才逐漸應用於生物膜之防治。本實驗以連續培養系統模擬白色念珠菌生物膜生長過程,再比較懸浮細胞與生物膜之亞甲基藍光動力抑菌劑量。以波長 630 ± 10 nm 之發光二極體照光 360 J/cm2,106 懸浮生菌需 2 nmol 亞甲基藍。而照光 480 J/cm2 時,108 懸浮生菌需 210 nmol;含 108 生菌數之生物膜所需劑量為1447 nmol。結果顯示生物膜所需劑量僅比懸浮細胞高出 7.23 倍。而 106 懸浮生菌於 5 μM 亞甲基藍中,分別照光 120 與 480 J/cm2,其菌落以三苯基四氮唑化氯 (TTC) 試驗,各可觀察到 6.6±4.1% 及 20.8±8.9% 粒線體受損之菌落。經光動力傷害之白色念珠菌粒線體以 JC-1 螢光染色後觀察,可發現其粒線體膜電位明顯下降,而死亡菌體則無法觀察到螢光反應。以螢光光度計檢測菌體內之 mitotracker green FM 及 JC-1 螢光強度發現,受光動力傷害之菌體,其螢光強度大幅下降,而死亡菌體則無明顯螢光反應。此研究顯示,亞甲基藍光動力抑制生物膜效果不受生物膜抗性機制之影響,可能是因為光動力作用直接傷害菌體之胞器,而不同於一般抗生素之抑制菌體生理活性。因此光動力作用在未來微生物防治上具有相當的潛力。

Photodynamic inactivation (PDI) of Candida albicans planktonic and biofilm cells by a phtotosensitizer, methylene blue (MB), has been investigated. For the planktonic and biofilm cells, MB inhibition efficiency of yeast was found to increase with both increasing MB concentration and light dose. But increasing the light dose could not further improve the antimicrobial activity, if the maximum excitation level was less attainable than the necessary minimum threshold. Complete inactivation can be achieved when the excitation level of MB is somewhere above the threshold. No viable cells were detected when 106 planktonic cells were exposed to 2 nmol MB and a light dose of 360 J/cm2, or 108 planktonic cells were exposed to 210 nmol MB and a light dose of 360 J/cm2 or 108 biofilms were exposed to 1447 nmol MB and a light dose of 360 J/cm2. Then, triphenyltetrazolium chloride (TTC)-agar was overlaid on the colonies formed from PDT treated cells, and several colonies with injury mitochondria were found. Dyeing the PDT-treated injury yeasts by JC-1 and observing them by fluorescence microscope, the potential of the mitochondria outer membrane decreased obviously. The MTG (mitotracker green FM) and JC-1 fluorescence intensity was detected by the fluorophotometer, the fluorescence intensity of the damaged cells decreased obviously, and no signal of the dead cells. This study shows that bilfilm resistance does not decrease MB-PDT of microbes. It is possible that PDT directly destroys organelles, instead of the physiological activity inhibition. Therefore, PDT is a potential way to prevent the microbes infection of in the future.

中文摘要....................................................................................III
英文摘要....................................................................................IV
壹、前言.....................................................................................1
1. 生物膜....................................................................................1
1.1 環境中之生物膜......................................................................1
1.2 生物膜之形成.........................................................................2
1.3 生物膜之結構.........................................................................5
1.4 生物膜之抗藥性機制................................................................7
1.5 白色念珠菌生物膜之研究..........................................................9
1.5.1 白色念珠菌之危害................................................................9
1.5.2 白色念珠菌生物膜之危害.....................................................12
1.5.3 白色念珠菌生物膜形成之環境因素.........................................13
1.5.4 白色念珠菌生物膜之生成過程...............................................15
1.6 生物膜之防治........................................................................17
2. 光動力作用.............................................................................18
2.1 光動力作用之歷史及應用........................................................18
2.2 光動力作用之機制.................................................................19
2.3 光動力作用之要素.................................................................21
2.3.1 光源.............................................................................21
2.3.2 光感物質............................................................................22
2.3.3 氧氣..................................................................................23
3. 亞甲基藍光動力研究之回顧.......................................................25
4. 氧化作用對粒線體之傷害..........................................................26
5. 研究動機及目的.......................................................................26
貳、材料與方法...........................................................................28
1. 菌種.......................................................................................28
2. 儀器.......................................................................................28
2.1 光動力作用裝置.....................................................................28
2.2 生物膜培養系統.....................................................................29
3. 實驗材料與方法.......................................................................30
3.1 磷酸緩衝液之配置..................................................................30
3.2 白色念珠菌懸浮菌體之培養與定量............................................30
3.3 白色念珠菌生物膜之培養與定量...............................................30
4. 光動力對白色念珠菌之抑制實驗.................................................31
4.1 光感物質之篩選.....................................................................31
4.2 PpIX 生合成之測定................................................................31
4.3 亞甲基藍對 106 CFU 懸浮菌體之光動力抑制.............................31
4.4 亞甲基藍對 108 CFU 懸浮菌體之光動力抑制.............................32
4.5 亞甲基藍對生物膜之光動力抑制...............................................32
5. 亞甲基藍激發量之測定..............................................................32
5.1 106 CFU 懸浮菌體.................................................................32
5.2 108 CFU 懸浮菌體.................................................................33
5.3 108 CFU 生物膜....................................................................33
6. 光動力作用對白色念珠菌粒線體損傷之測定..................................33
6.1 以 TTC 評估對於粒線體損傷....................................................33
6.2 JC-1 對於粒線體膜電位之測定.................................................34
6.2.1 以螢光顯微鏡觀察粒線體電位變化.........................................34
6.2.2 以螢光光度計偵測粒線體電位...............................................35
6.3 以 Mitotracker* Green FM (MTG) 測粒線體膜結構...................35
參、結果....................................................................................36
1. 光感物質之篩選......................................................................36
1.1 部花青素 (merocyanine 540, MC 540)...................................36
1.2 靛氰綠 (indocyanine green, ICG)...........................................37
1.3 胺基酮戊酸 (delta-aminolevulinic acid, δ-ALA).....................38
1.3.1 δ-ALA 之光動力抑制效果....................................................38
1.3.2 PpIX 生合成之測定..............................................................39
1.4 亞甲基藍 (methylene blue, MB)..............................................41
2. 白色念珠菌生物膜生成曲線........................................................42
3. 亞甲基藍之光動力抑制..............................................................44
3.1 對懸浮細胞 (106 CFU) 之抑制作用...........................................44
3.2 對懸浮細胞 (108 CFU) 之抑制作用...........................................46
3.3 對生物膜 (108 CFU) 之抑制作用..............................................48
4. TTC 對於光動力造成粒線體損傷之評估.......................................50
5. JC-1 對於粒線體電位改變之觀察................................................51
5.1 以螢光顯微鏡觀察粒線體電位...................................................51
5.2 以螢光光度計偵測粒線體電位...................................................53
5.2 以螢光光度計偵測粒線體功能及構造.........................................54
肆. 討論......................................................................................55
1. Pp IX 生合成的測定..................................................................55
2. 亞甲基藍之光動力抑制效果........................................................55
3. 亞甲基藍激發量與抑菌能力之關係...............................................56
4. 光動力作用對於粒線體之傷害.....................................................58
伍、結論及未來之展望...................................................................60
陸、參考文獻................................................................................61

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