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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃慶璨 | |
dc.contributor.author | Ya-Ping Tu | en |
dc.contributor.author | 凃雅屏 | zh_TW |
dc.date.accessioned | 2021-06-13T06:43:17Z | - |
dc.date.available | 2008-08-01 | |
dc.date.copyright | 2005-08-01 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-29 | |
dc.identifier.citation | 伍、參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35182 | - |
dc.description.abstract | 光動力治療(Photodynamic therapy, PDT) 是一種使用特定波長
之激發光將光感物質(photosensitizer) 激發成高能量激發態,經由能 量或電子傳遞,將細胞周圍氧分子轉變成有細胞毒性的單態氧 (1O2),或與環境分子作用產生自由基,造成細胞死亡的方法,目前已 應用於癌症治療及微生物防治(Photodynamic inactivation, PDI)。研究 顯示,將光動力治療應用在微生物防治上已有部分成效,而積極開發 新光源及光感物質,使光動力治療能更有效發揮,為目前研究的新趨 勢。藻藍素(phycocyanin, PC) 為螺旋藻(Spirulina platensis) 中的一 種水溶性螢光蛋白,可吸收光能並進行能量及電子傳遞,具有光動力 治療之潛力。本實驗由螺旋藻Spirulina platensis 中萃取藻藍素作為光 感物質,並以發光二極體(Light emitting diodes,LED) 為光源,對 革蘭氏陽性及陰性菌進行光動力抑制,並進一步探討菌體在懸浮狀態 與生物膜狀態下,光動力殺菌效果是否不同。實驗結果顯示由螺旋藻 中萃取並純化出藻藍素,回收率可達56.1%,純度(A620nm/280nm) 為 4.56,其品質與商品化之藻藍素相當。而以藻藍素進行光動力治療, 在藻藍素濃度100 µg ml-1,照光強度360 J cm-2 之條件下,可將濃度 107 CFU ml-1 之革蘭氏陽性菌S. aureus 及S. epidermis 懸浮細胞完全 抑制,但對革蘭氏陰性菌E. coli 及P. aeruginosa 之效果則不顯著; 而以藻藍素對S. aureus 及S. epidermis 生物膜進行光動力抑制,在藻 藍素濃度900 µg ml-1,照光強度360 J cm-2 之條件下。亦可將108 CFU cm-2 之生物膜菌體完全殺滅。此研究顯示由螺旋藻中萃取之天然光感 II 物質藻藍素對革蘭氏陽性菌之懸浮菌體及生物膜細胞皆有良好之光 動力抑制效果,在未來的應用上極具潛力。 關鍵詞:光動力抑制、螺旋藻、藻藍素、生物膜 | zh_TW |
dc.description.abstract | Photodynamic inactivation (PDI) utilized photosensitizers and light
with appropriate wave length to give a phototoxic response, normally via oxidative damage. Although PDI might be an effective approach in antimicrobial treatment, no photosensitizer is suitable for all possible applications. Therefore, the development of new photosensitizers became important to overcome the shortage of PDI. Phycocyanin (PC), a water-soluble non-toxic biliprotein, is one of the major constituents of Spirulina platensis. The photobiological properties of PC suggested its possibility to be a photosensitizer in photodynamic therapy. The goal of this study was to investigate the effect of the PC extracted from Sp. platensis on photodynamic inactivation against bacterial planktonic and biofilm cells. The extraction and purification of PC was accomplished by fractional precipitation with ammonium sulfate precipitation, following by ion-exchange chromagraphy on Macro-Prep DEAE Support system. The purity of PC was examined by absorbance and fluorescence spectrometry. Both Gram-positive and Gram-negative bacteria were tested for PC-PDI. Pure PC was finally obtained from Sp. platensis with purity ratio (A620nm/280nm) 4.56 and recovery rate 56.1%. After PC-PDI treatment, which carried with 100 µg ml-1 of PC and irradiation light dose of 360 J cm-2, no viable cell of the Gram-positive Staphylococcus aureus and S. epidermis planktonic cells were detected. However, the Gram-negative Escherichia coli and Pseudomonas aeruginosa were resistant to the PC-PDI treatment. Experiments with PC-PDI against S. aureus and S. epidermis biofilms showed that both species of the tested biofilms were sensitive to the PC-PDI treatment, with a decrease of about 8-log of viable cells. This study showed that the PC from Sp. platensis was a potential photosensitizer to inactivated Gram-positive bacteria planktonic cells and biofilms. Keyword: Photodynamic Inactivation, Spirulina platensis, Phycocyanin, Biofilm | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:43:17Z (GMT). No. of bitstreams: 1 ntu-94-R92b47403-1.pdf: 1831956 bytes, checksum: ca603794810a9622f8b730cb8ec5444d (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 目錄
中文摘要------------------------------------------------------------------------- I 英文摘要----------------------------------------------------------------------- III 目錄------------------------------------------------------------------------------- i 表次------------------------------------------------------------------------------- v 圖次------------------------------------------------------------------------------ vi 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 1.4.1 物理屏障-------------------------------------------------------- 7 1.4.2 生理適應-------------------------------------------------------- 7 1.4.3 細胞對細胞的聯繫-------------------------------------------- 7 1.5 生物膜防治------------------------------------------------------------ 8 2. 光動力抑制------------------------------------------------------------------ 9 2.1 光動力作用之歷史與應用------------------------------------------ 9 2.2 光動力作用之機制------------------------------------------------- 10 2.3 光動力作用之要素------------------------------------------------- 15 2.3.1 光源------------------------------------------------------------ 15 2.3.2 光感物質------------------------------------------------------ 17 2.3.3 氧氣------------------------------------------------------------ 18 ii 2.4 光感物質之開發---------------------------------------------------- 19 2.5 天然光感物質------------------------------------------------------- 21 3. 藻藍素---------------------------------------------------------------------- 22 3.1 螺旋藻之經濟價值------------------------------------------------- 22 3.2 藻藍素在生物體之角色------------------------------------------- 23 3.3 藻藍素之醫療價值------------------------------------------------- 26 3.4 藻藍素之光感性質及潛力---------------------------------------- 29 3.5 萃取方法之研究回顧---------------------------------------------- 31 4. 動機與目的---------------------------------------------------------------- 32 貳、材料與方法---------------------------------------------------------------- 34 1 菌種------------------------------------------------------------------------- 34 2 儀器------------------------------------------------------------------------- 34 2.1 藻藍素純化---------------------------------------------------------- 34 2.2 生物膜培養系統---------------------------------------------------- 34 2.3 光動力作用裝置---------------------------------------------------- 36 3 實驗材料與方法---------------------------------------------------------- 37 3.1 緩衝溶液之配製---------------------------------------------------- 37 3.1.1 磷酸鉀緩衝溶液之配製------------------------------------ 37 3.1.2 等張磷酸鹽緩衝溶液之配製------------------------------ 37 3.2 懸浮菌體培養與定量---------------------------------------------- 37 3.2.1 金黃色葡萄球菌之培養------------------------------------ 37 3.2.2 白色表皮葡萄球菌之培養--------------------------------- 38 3.2.3 大腸桿菌之培養--------------------------------------------- 38 3.2.4 綠膿桿菌之培養--------------------------------------------- 38 iii 3.2.5 活菌數定量方法--------------------------------------------- 38 3.3 生物膜培養與定量------------------------------------------------- 39 3.3.1 S. aureus 與S. epidermis 生物膜之培養------------------ 39 3.3.2 生物膜之定量------------------------------------------------ 39 4 藻藍素純化與分析------------------------------------------------------- 39 4.1 藻藍素粗萃取------------------------------------------------------- 39 4.2 藻藍素純化---------------------------------------------------------- 40 4.3 藻藍素定性分析---------------------------------------------------- 40 4.3.1 聚丙烯醯胺膠體電泳--------------------------------------- 40 4.3.2 液相層析質譜儀分子量決定法--------------------------- 40 4.3.3 測定藻藍素之光譜特性分析------------------------------ 40 4.4 藻藍素定量分析---------------------------------------------------- 41 4.4.1 蛋白質定量法------------------------------------------------ 41 4.4.2 藻藍素吸收光譜分析--------------------------------------- 41 4.4.3 藻藍素放射光譜分析--------------------------------------- 41 5 藻藍素之光動力抑制實驗---------------------------------------------- 42 5.1 藻藍素對懸浮菌體之光動力抑制------------------------------- 42 5.2 藻藍素對生物膜之光動力抑制---------------------------------- 42 参、結果與討論---------------------------------------------------------------- 43 1. 生物膜培養與定量------------------------------------------------------- 43 1.1 金黃色葡萄球菌生物膜之生長曲線---------------------------- 43 1.2 白色表皮葡萄球菌生物膜之生長曲線------------------------- 45 2. 藻藍素萃取與純化------------------------------------------------------- 46 2.1 萃取與純化條件---------------------------------------------------- 46 iv 2.1.1 硫酸銨沈澱法濃度條件測試與結果--------------------- 46 2.1.2 Rivanol 處理法之比較-------------------------------------- 47 2.1.3 藻藍素純化結果--------------------------------------------- 49 2.2 藻藍素定性分析---------------------------------------------------- 50 2.2.1 藻藍素分子量------------------------------------------------ 50 2.2.2 藻藍素之吸收光譜------------------------------------------ 52 2.2.3 藻藍素之放射光譜------------------------------------------ 53 2.3 藻藍素定量分析---------------------------------------------------- 55 3. 藻藍素之光動力抑制實驗---------------------------------------------- 58 3.1 藻藍素對懸浮菌體之光動力抑制作用------------------------- 58 3.1.1 革蘭氏陽性菌------------------------------------------------ 58 3.1.2 革蘭氏陰性菌------------------------------------------------ 64 3.2 藻藍素對生物膜之光動力抑制作用---------------------------- 67 3.2.1 金黃色葡萄球菌生物膜------------------------------------ 67 3.2.2 白色表皮葡萄球菌生物膜--------------------------------- 69 4. 討論------------------------------------------------------------------------- 70 4.1 藻藍素萃取與純化------------------------------------------------- 70 4.2 藻藍素之光動力抑制效果---------------------------------------- 71 4.3 藻藍素之光動力作用對革蘭氏陽性菌與陰性菌之差別---- 74 4.4 藻藍素之光動力作用對懸浮細胞與生物膜之抑制效果---- 77 肆、結論與未來展望---------------------------------------------------------- 78 伍、參考文獻------------------------------------------------------------------- 79 表次 表一、藻藍素基本性質------------------------------------------------------- 30 表二、液相層析質譜儀分析結果------------------------------------------- 51 表三、藻藍素萃取與純化結果---------------------------------------------- 57 表四、純化結果與前人研究之比較---------------------------------------- 70 表五、藻藍素之光動力抑制結果與前人研究之比較------------------- 73 圖次 圖一、生物膜之形成示意圖-------------------------------------------------- 4 圖二、生物膜之蕈狀結構假想圖-------------------------------------------- 7 圖三、光動力作用機制------------------------------------------------------- 12 圖四、光動力作用之能量轉移---------------------------------------------- 13 圖五、光感物質與環境分子間之作用------------------------------------- 14 圖六、藻膽體構造------------------------------------------------------------- 24 圖七、不同色素之吸收光譜------------------------------------------------- 25 圖八、藻藍素之發色團------------------------------------------------------- 27 圖九、藻藍素結構------------------------------------------------------------- 28 圖十、生物膜反應器及裝置------------------------------------------------- 35 圖十一、紅色發光二極體---------------------------------------------------- 36 圖十二、金黃色葡萄球菌生物膜之生長曲線---------------------------- 44 圖十三、白色表皮葡萄球菌生物膜之生長曲線------------------------- 45 圖十四、藻藍素粗萃取液經不同濃度之硫酸銨沈澱結果------------- 47 圖十五、兩種不同萃取方式------------------------------------------------- 48 圖十六、純化所得之藻藍素------------------------------------------------- 49 圖十七、13% 聚丙烯醯胺膠體電泳結果--------------------------------- 51 圖十八、藻藍素吸收光譜---------------------------------------------------- 52 圖十九、藻藍素放射光譜---------------------------------------------------- 53 圖二十、不同濃度藻藍素之放射光譜------------------------------------- 54 圖二十一、濃度與光譜強度之線性關係---------------------------------- 56 圖二十二、以藻藍素標準品對革蘭氏陽性菌S. aureus 懸浮細胞進行 vii 光動力抑制----------------------------------------------------- 60 圖二十三、以藻藍素標準品對革蘭氏陽性菌S. epidermis 懸浮細胞進 行光動力抑制-------------------------------------------------- 61 圖二十四、以藻藍素樣品對革蘭氏陽性菌S. aureus 懸浮細胞進行光 動力抑制-------------------------------------------------------- 62 圖二十五、以藻藍素樣品對革蘭氏陽性菌S. epidermis 懸浮細胞進行 光動力抑制----------------------------------------------------- 63 圖二十六、以藻藍素標準品對革蘭氏陰性菌P. aeruginosa 懸浮細胞進 行光動力抑制-------------------------------------------------- 65 圖二十七、以藻藍素標準品對革蘭氏陰性菌E. coli 懸浮細胞進行光動 力抑制----------------------------------------------------------- 66 圖二十八、以藻藍素樣品對S. aureus 生物膜進行光動力抑制------- 68 圖二十九、以藻藍素樣品對S. epidermis 生物膜進行光動力抑制--- 69 圖三十、藻藍素激發量與菌株之關係------------------------------------- 75 圖三十一、菌體周圍之藻藍素濃度---------------------------------------- 76 | |
dc.language.iso | zh-TW | |
dc.title | 以螺旋藻萃取之C-藻藍素對細菌懸浮細胞與生物膜之
光動力抑制作用 | zh_TW |
dc.title | Photodynamic Inactivation against Bacterial Planktonic Cells and Biofilms with C-Phycocyanin Extracted from Spirulina platensis | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許瑞祥,陳進庭 | |
dc.subject.keyword | 光動力抑制,螺旋藻,藻藍素,生物膜, | zh_TW |
dc.subject.keyword | Photodynamic Inactivation,Spirulina platensis,Phycocyanin,Biofilm, | en |
dc.relation.page | 87 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-30 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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