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研究生:李佳芬
研究生(外文):Chia-Fen Lee
論文名稱:以部花青素褐藻酸鈣晶球進行光動力作用處理流動狀態之金黃色葡萄球菌
論文名稱(外文):Inactivation of Flowing Staphylococcus aureus by Photodynamic Antimicrobial Chemotherapy with Merocyanine 540 in Alginate Beads
指導教授:黃慶璨
指導教授(外文):Ching-Tsan Huang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:82
中文關鍵詞:MRSA固定化技術發光二極體光動力治療
外文關鍵詞:LEDlight emitting diodemethicillin-resistant Staphylococcus aureusphotodynamic therapyimmobilizationMRSA
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由於抗生素的濫用,細菌抗藥性已成為世界共通問題,發展新的微生物防治法更是當務之急。光動力作用(Photodynamic action)是利用特定波長的光源來激發光感物質,不穩定的激發態將其能量轉換給周旁的氧分子後,進一步氧化細胞生物分子以造成破壞。早期光動力研究多著眼於癌細胞破壞,近年研究指出,此一機制在殺滅病毒、細菌乃至於真菌,亦有相當成效。然而,前人以光動力作用處理微生物多著眼於體外實驗,以不同光源激發各種光感物質來達到抗菌效果,或修飾光感物質的結構來加強與菌體間的親合性。近年來已有少數體內的局部測試,但在克服懸浮狀態下光動力作用對正常細胞的破壞仍有其瓶頸。此外,雖然已有研究選擇部花青素作為光感物質來處理微生物,但以細菌作為實驗材料仍屬少數。本實驗嘗試以褐藻酸鈣包埋部花青素以進行光動力作用處理Staphylococcus aureus與methicillin-resistant S. aureus (MRSA),藉由部花青素藻膠晶球存在的孔隙來區隔細菌與正常血球細胞。結果顯示藻膠晶球直徑界於1.81-3.10 mm之間,取十顆晶球與濃度108 CFU/mL的菌液共振盪十分鐘後,約有1/10之菌數進入晶球。在光能量72 J/cm2的處理下,無論以部花青素原液50或100 �慊/mL製備的晶球,皆可達到完全抗菌的效果;以循環反應器系統處理菌液濃度108 CFU/mL的野生株和抗藥性菌株,其光動力抗菌最大效果約可達5 log CFU/mL。在血液測試上,抗菌效果雖不如以緩衝液處理時顯著,但紅血球仍可保持完整。此研究顯示出本系統在滅殺流動狀態的微生物的應用上,具有相當的潛力。
Photodynamic antimicrobial chemotherapy (PACT), derived from photodynamic therapy (PDT), utilized photosensitizer (PS) and visible or ultraviolet light in order to give a phototoxic response, normally via oxidative damage to microorganisms. Because neither of the two major factors (lights and PSs) of the PACT is inherently bactericidal, it could be a useful and simple way to treat and control antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Although lethal photosensitization of a wide range of microorganisms has been reported, the lack of specificity and inefficiency of light activation limit its application. The goal of this study is to establish a PACT system to treat flowing S. aureus and MRSA by immobilized PS that could be irradiated in vitro. Merocyanine 540 (MC540), a fluorescent dye that binds to the outer surface of the plasma membrane, had been widely used in blood sterilization and in inactivation against both planktonic culture and biofilm of S. aureus. In order to distinguish blood cells and bacteria, MC540 was immobilized by alginate beads to form porous medium which only allowed bacteria passed through. The characteristics of beads and the ability of MC540-alginate beads irradiated by green light emitting diode (LED) to inactivation flowing S. aureus strains in the circular reactor were reported. In addition, a blood sample containing S. aureus was also treated using this approach. The diameter of beads was between 1.81 and 3.10 mm. After 108 CFU/mL bacteria incubated with 10 alginate beads, there was around 1/10 bacteria bound to beads. In small scale treatment, when incident dose was up to 72 J/cm2, no viable cell of S. aureus was could be detected. In circular reactor, the best result of PACT treatment could suppress around 5 log CFU/mL. Although PACT showed minor inactivation in blood, this research showed a potential strategy of inactivation bacteria in blood and proposed a novel strategy by immobilizing photosensitizer to enhance PACT. This system was efficient to damage both wild type and antibiotic-resistant S. aureus.
目錄
頁次
中文摘要 ----------------------------------------------------------------- I
英文摘要 ----------------------------------------------------------------- II
第一章、緒論 ----------------------------------------------------------- 1
1. 微生物防治 ------------------------------------------------------- 1
1.1 微生物與疾病 ------------------------------------------------ 1
1.2 抗生素治療簡史與發展瓶頸 ------------------------------ 2
2. 光動力治療 ------------------------------------------------------- 7
2.1 發展歷史 ------------------------------------------------------ 7
2.2 作用機制 ------------------------------------------------------ 8
2.3 作用要素 ------------------------------------------------------ 10
2.3.1 光源 ------------------------------------------------------- 10
2.3.1.1雷射 -------------------------------------------------- 10
2.3.1.2 非雷射 ----------------------------------------------- 10
2.3.2 光感物質 ------------------------------------------------- 11
2.3.2.1 基本特性 -------------------------------------------- 11
2.3.2.2 部花青素 -------------------------------------------- 12
2.4相關應用 ------------------------------------------------------ 14
2.5 微生物防治之潛力 ------------------------------------------ 14
2.5.1 研究回顧 ------------------------------------------------- 14
2.5.2 發展要點 ------------------------------------------------- 16
2.5.2.1 專一性 ----------------------------------------------- 16
2.5.2.2 局部性 ----------------------------------------------- 17
3. 褐藻膠之固定化技術 ------------------------------------------- 19
3.1 固定化技術 ---------------------------------------------------
3.2 褐藻膠之結構與凝膠性質 --------------------------------- 19
24
3.3 褐藻膠之應用 ------------------------------------------------ 25
4. 本研究之動機與目的 ------------------------------------------- 28
第二章、材料與方法 -------------------------------------------------- 30
1. 菌株 ---------------------------------------------------------------- 30
2. 實驗材料與方法 ------------------------------------------------- 30
2.1 循環反應器系統 --------------------------------------------- 32
2.2 菌體培養 ------------------------------------------------------ 32
2.3 藥劑配製 ------------------------------------------------------ 32
2.3.1等張磷酸鹽緩衝溶液之配製 ------------------------- 32
2.3.2部花青素母液之配製 ---------------------------------- 34
2.4 褐藻酸鈣晶球之製作 --------------------------------------- 34
2.5 部花青素褐藻酸鈣晶球之製作 --------------------------- 34
3. 褐藻酸鈣晶球性質分析 ---------------------------------------- 35
3.1 外觀與粒徑 --------------------------------------------------- 35
3.1.1 解剖顯微鏡 ---------------------------------------------- 35
3.1.2 冷凍包埋切片 ------------------------------------------- 35
3.2 水分含量測定 ------------------------------------------------ 36
3.3 褐藻酸鈣晶球作用後菌體之計數 ------------------------ 36
3.4 褐藻酸鈣晶球作用後菌體之分布 ------------------------ 37
4. 部花青素褐藻酸鈣晶球性質分析 ---------------------------- 38
4.1 部花青素包埋率 --------------------------------------------- 38
4.2 部花青素與褐藻酸釋出率 --------------------------------- 38
4.2.1 部花青素定量 ------------------------------------------- 38
4.2.2 褐藻酸定量 ---------------------------------------------- 39
4.3 儲存安定性 --------------------------------------------------- 39
5. 部花青素褐藻酸鈣晶球於緩衝液之光動力抗菌實驗 ---- 39
5.1 小規模微量離心管之實驗 --------------------------------- 39
5.2 循環反應器系統之實驗 ------------------------------------ 40
5.2.1 部花青素褐藻酸鈣晶球之光動力抗菌效果 ------- 40
5.2.2 部花青素褐藻酸鈣晶球光分解產物抗菌效果 ---- 40
6. 部花青素褐藻酸鈣晶球於血液之光動力抗菌實驗 ------- 42
6.1 血球定量 ------------------------------------------------------ 42
6.2 循環反應器系統之實驗 ------------------------------------ 42
7. 統計分析 ---------------------------------------------------------- 43
第三章、結果與討論 -------------------------------------------------- 44
1. 褐藻酸鈣晶球性質分析 ---------------------------------------- 44
1.1 外觀與粒徑 --------------------------------------------------- 44
1.2 水分含量變化 ------------------------------------------------ 45
1.3 作用後菌體之計數 ------------------------------------------ 45
1.4作用後菌體之分布 ------------------------------------------ 48
2. 部花青素褐藻酸鈣晶球濃度測定 ---------------------------- 52
2.1 部花青素包埋率 --------------------------------------------- 52
2.2 部花青素與褐藻酸釋出率 --------------------------------- 52
2.3儲存安定性 --------------------------------------------------- 55
3. 部花青素褐藻酸鈣晶球於緩衝液之光動力抗菌 ---------- 55
3.1 野生株 --------------------------------------------------------- 55
3.2 抗藥性菌株 --------------------------------------------------- 57
4. 部花青素褐藻酸鈣晶球於血液之光動力抗菌 ------------- 61
5. 部花青素褐藻酸鈣晶球光分解產物抗菌效果 ------------- 66
6. 不同處理之部花青素褐藻酸鈣晶球冷凍包埋切片 ------- 67
7. 循環反應器系統之設計 ---------------------------------------- 70
第四章、結論 ----------------------------------------------------------- 72
第五章、未來研究方針 ----------------------------------------------- 73
第六章、參考文獻 ----------------------------------------------------- 74
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