臺灣博碩士論文加值系統

光動力療法（photodynamic therapy, PDT）乃是結合感光劑及適當波長之光源，經由電子或能量轉移產生能進行無特異性目標攻擊之活性氧類（reactive oxygen species），以破壞腫瘤及病原體。臨床上已使用五胺基酮戊酸（5-aminolevulinic acid, ALA）作為感光劑前驅物，針對表面傷口感染進行治療。多數病人之傷口皆能有效治癒復原，但亦有少數病人無法有效抑制細菌感染。本研究即針對四種由兩位分別接受一次和五次ALA-PDT之病人所分離而得之細菌－oxacillin-resistant Staphylococcus aureus（ORSA）, Enterococcus faecalis, Pseudomonas aeruginosa, Shewanella putrefaciens－於體外使用ALA進行光動力抑菌試驗（ALA-PDI）。我們以隨機擴增片段多型性（RAPD）方法將屬同一菌種之菌株加以分型，發現各菌種之菌株含有至少兩種以上的RAPD型別。我們接著測定各菌種之菌株對ALA-PDI之感受性，並以fluorometry測定其代謝ALA所產生具感光性之紫質衍生物的量，發現了不同菌種對ALA-PDI即有不同之反應。具不同型別的P. aeruginosa對ALA-PDI及其菌體內代謝ALA所產生的紫質衍生物之量均有劑量依存關係。不同型別的ORSA菌株則在ALA濃度約0.1 mM時表現最高的感受性，同時其菌體內所產生之porphyrin derivatives的量亦達到最高。E. faecalis菌株則對ALA-PDI無明顯反應，且其菌體內亦偵測不到紫質衍生物的產生，推測可能該菌無法有效攝取或利用ALA合成紫質衍生物而使ALA-PDI無效。較令人困惑的是，ALA-PDI對S. putrefaciens雖無殺菌效果，但我們仍能於此菌之菌體內偵測到大量紫質衍生物的產生。本研究之結果顯示不同細菌對ALA-PDI之感受度即有差異，因此，臨床上採用ALA-PDT治療細菌感染前，須先試驗各個菌株對ALA-PDI之感受性，找出最適當之感光劑濃度及光劑量，方能將光動力療法之殺菌能力發揮至最大。

　　Photodynamic therapy （PDT） combines the photosensitizers （PSs） and visible light to produce a phototoxic response that results in oxidative damages to a variety of targets including nucleic acids, proteins, and lipids. It has been noticed that bacterial strains of the same species were repeatedly isolated from the wounds of two patients who received one and five, respectively, courses of PDT with 5-aminolevulinic acid （ALA）, a precursor of photoactive porphyrin derivatives. From one of them the oxacillin-resistant Staphylococcus aureus （ORSA）, Enterococcus faecalis and Shewanella putrefaciens were isolated, while from the other patient only Pseudomonas aeruginosa was isolated. The strains of each species exhibited at least two different randomly amplified polymorphic DNA （RAPD） types. We then examined the strains of different RAPD types for their susceptibility to photodynamic inactivation（PDI） with ALA and the amount of porphyrins produced in their cells. We found that strains of ORSA and P. aeruginosa could be killed by ALA-PDI but those of E. faecalis and S. putrefaciens could not. Both the susceptibility to ALA-PDI and amounts of porphyrins synthesized from ALA showed a dose-dependent response for the P. aeruginosa strains. Strains of ORSA showed an optimal response to 0.1 mM of ALA for both the sensitivity to ALA-PDI and porphyrins production from ALA. No fluorescence representing the porphyrins produced from ALA was detected in E. faecalis, suggesting that this species may be unable to uptake or metabolize ALA to porphyrins, which leads to irresponsiveness of this bacterial species to ALA-PDI. Intriguingly, although strong fluorescence signals were detected in the cells of S. putrefaciens, strains of this species were resistant to ALA-PDI. These results suggest that the bacterial pathogens may not be equally sensitive to PDT with ALA, and it may be necessary to test each isolate to find the optimal ALA-PDT conditions.

中文摘要 i
英文摘要 ii
謝誌 iii
目錄 iv
表目錄 vi
圖目錄 vii
符號與縮寫 ix
緒論 1
　一、光動力治療法 1
　二、光動力學治療法於非感染性疾病之應用 2
　三、光動力治療法於感染性疾病之應用 3
　四、感光劑前驅物五胺基酮戊酸 3
　五、以五胺基酮戊酸作為感光劑前驅物對久治不癒的傷口進行光動力療法
　　　 5
　六。Oxacillin-resistant Staphylococcus aureus, Enterococcus faecalis, Pseudo- monas aeruginosa, 及Shewanella putrefaciens四菌種之簡介 6
研究目的 9
材料與方法 11
　一、實驗菌種與保存方式 11
　　１.實驗菌種 11
　　２.細菌培養液及培養盤之配製 11
　　３.菌種的培養與保存方法 12
　二、Randomly amplified polymorphic DNA（RAPD）技術 13
　　１.小量純化染色體DNA的方法 13
　　２.聚合酶連鎖反應（Polymerase Chain Reaction, PCR） 14
　　３.DNA電泳分析（DNA gel electrophoresis） 18
　三、光動力抑菌實驗（Photodynamic inactivation） 19
　　１.細菌生長曲線及菌量評估之建立 19
　　２.光動力抑菌實驗 19
　　３.統計方法 21
　四、紫質衍生物之偵測 21
　　１.檢體之製備 21
　　２.螢光物質偵測 22
研究結果 23
　一、利用RAPD方法比較菌株之異同 23
　二、以ALA作為感光劑前驅物的光動力法對臨床菌株之殺菌效果的評估
24
　三、細菌代謝ALA產生porphyrin derivatives之定性分析 27
討論 30
結論 36
未來展望 37
參考文獻 38
表 48
圖 51
附錄 72
自述 75

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