臺灣博碩士論文加值系統

光動力療法(Photodynamic treatment)，簡稱PDT，是感光劑在光線及氧氣充足的環境下產生可毒殺癌細胞及微生物的一種反應，許多感光劑的作用機制是透過與核酸之高親合力，在受到光線激發下產生自由基，破壞微生物結構並會造成蛋白質及核酸之交叉結合。在本研究中，我們探討最廣泛使用之感光劑甲基藍，是否對腸病毒71型有光動力去活化作用。腸病毒71型屬於小RNA病毒，可造成幼兒嚴重中樞神經感染，目前無有效的抗病毒藥物及疫苗可供使用，一般認為病毒透過糞口途徑傳播，對於酒精消毒則具有抗性。我們證明了甲基藍在可見光的激發下，有效地使固體表面上之腸病毒71型去活化而達到消毒效果。病毒懸浮狀態下，對於甲基藍光動力效應具有濃度及光強度依存性，而單獨給予甲基藍處理、單獨光線照射或是先將甲基藍光照後再處理病毒，均無光動力去活化作用產生。在懸浮狀態下，對於腸病毒71型最佳的光動力條件為光強度200 J/cm2及甲基藍濃度0.1 mM，此條件並可有效去活化其他腸病毒屬病毒，包括小兒麻痺病毒1型、克沙奇病毒A2、A3、A16及B3。在西方墨點及反轉錄聚合酶鏈反應分析中亦發現光動力效應會破壞病毒蛋白及RNA基因體，可能為PDT抑制腸病毒71型之分子機制。因此，光動力效應可作為清除環境中腸病毒71型的一種消毒方法，藉此減少腸病毒傳播降低感染率。

Photodynamic treatment (PDT) has been applied to destroy cancer cells and a variety of microorganisms including bacteria and viruses by combining light and photosensitizing agents in an oxygen-rich environment. Certain photosensitizing agents can bind strongly with nucleic acids and generate reactive oxygen species after activation upon illumination, which is responsible for the disruption of viral structures and viral nucleic acid-protein cross-linkage. In this study, we have tested whether methylene blue (MB), the most widely used photosensitizing agent, is feasible for photodynamic inactivation of enterovirus 71 (EV71), a non-enveloped virus belonging to the Picornaviridae family, which can cause severe central nervous system infections and has neither anti-viral drug nor vaccine available. EV71 is transmitted through oral-fecal route and resistant to 70 % alcohol disinfection. We demonstrated that EV71 on solid surface could be photodynamically inactivated by illumination with visible light in the presence of MB. Photodynamic inactivation of EV71 in suspensions by MB showed dose- and light-intensity-dependent manners. Illumination without MB or MB alone in the dark and preactivation of MB showed no such effect. The optimal condition for photoinactivating EV71 required a light intensity of 200 J/cm2 and MB of 0.1 mM. This PDT condition could also inactivate other enteroviruses, including poliovirus 1, cosackievirus A2, A3, A16, and B3. Western blot and reverse-transcriptase PCR analysis indicated that the viral proteins and genome were broken after PDT. Thus, protein and genome damage appears to be involved in photoinactivation of EV71. In conclusion, PDT can inactivate EV71 efficiently and it may provide a method of eliminating environmental contaminated source of EV71 to prevent EV71 infections.

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VI
第一章 緒論 1
一、光動力效應 1
二、光動力效應抑制腫瘤相關研究 2
三、光動力效應抑制微生物相關研究 2
四、光動力效應之血液製劑消毒作用 3
五、甲基藍化合物特性及其光化學反應 4
六、腸病毒71型流行病學及防治 5
第二章 研究目的及特異目標 7
第三章 材料及方法 9
一、材料 9
1、細胞株 9
2、病毒 9
3、實驗動物 9
4、試劑套組 9
5、耗材 9
6、儀器 10
二、方法 11
1、細胞及病毒培養 11
2、甲基藍配製 12
3、固體表面腸病毒71型於不同相對濕度環境之存活率 12
4、固體表面腸病毒71型之光動力效應進行 12
5、懸浮狀態腸病毒71型之光動力效應進行 13
6、光動力效應處理之腸病毒71型在實驗動物感染力 13
7、甲基藍與病毒培養時間及光通量對於光動力效應影響 14
8、光動力效應處理腸病毒屬中其他病毒 14
9、西方墨點法 (Western blot) 15
10、反轉錄聚合酶鏈反應 (Reverse-trascriptase PCR, RT-PCR) 16
11、統計分析 18
第四章 結果 19
一、光動力效應去活化固體表面上之腸病毒71型 19
二、光動力去活化腸病毒71型呈現光能量及甲基藍劑量依存性 19
三、光動力處理下存活之腸病毒71型仍保持其致病性 20
四、甲基藍與病毒培養時間及光通量非主要光動力效應參數 20
五、光動力效應可去活化其他腸病毒屬病毒 21
六、甲基藍透過破壞腸病毒71型蛋白及RNA基因體進行光動力去活化 21
第五章 討論 23
參考文獻 28

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