棘阿米巴角膜炎是致病性棘阿米巴經由角膜外傷感染角膜的疾病，長期使用隱形眼鏡的人，角膜較易受損，因而有較大的風險受到感染。成大醫院眼科團隊從感染棘阿米巴角膜炎的病患身上分離出臨床株 (NCKH_A~NCKH_Q) 共17株，其中有部分病患為治療執行手術置換眼角膜或眼球摘除。聚六亞甲基雙胍鹽酸鹽簡稱PHMB，常用於治療棘阿米巴角膜炎。現今，出現PHMB藥物治療失敗之病例，因此須深入探究棘阿米巴感染及治療方式更是刻不容緩。本論文選用是品種、基因型以及可能的感染途徑皆相同的NCKH_B與NCKH_D進行實驗，測試ATCC標準株 (30010)、NCKH_B及NCKH_D對於100µg/ml PHMB的藥物敏感程度。以PHMB作用一小時後，標準株細胞95%死亡，而NCKH_B和NCKH_D的死亡率只有70%與60%。同時，我們觀察到NCKH_B與NCKH_D部分存活的蟲株會轉型成為偽囊體，尤其是NCKH_B存活的蟲株大部分皆轉變為偽囊體，纖維素合成酶-棘阿米巴轉型之關鍵酵素進行驗證。且利用聚合酶連鎖反應證實，而將藥物移除一小時後，可觀察到NCKH_D存活的蟲株部分已恢復其行動能力，而NCKH_B卻依然保持偽囊體的狀態。綜合以上結果，我們推測NCKH_B是利用轉變型態逃脫藥物毒殺；NCKH_D營養體擁有較高耐受性。為了提高治療成效，我們嘗試合併PHMB與轉錄抑制劑毒殺棘阿米巴，也證實此舉能達到加成的效果。此外，為了釐清蟲株對組織的侵犯能力，我們建立體外感染實驗平台，以臨床蟲株感染人類角膜，其後進行切片染色觀察，發現臨床蟲株皆具有感染人類及大鼠角膜的能力。總結以上實驗結果，我們已成功建立棘阿米巴的藥物測試及感染實驗平台，可提供使用於新藥治療成效測試，並期望合併藥物最終可達到臨床應用。

In this study, we isolated seventeen Acanthamoeba isolates (NCKH_A to NCKH_Q) associated with keratitis infection with various drug tolerances and disease severity. These isolates were identified by genus-specific PCR and sequencing. We used 100µg/ml PHMB to examine the drug sensitivity of these isolates, and observed that 95% of the cells belonging to the standard strain ATCC 30010 were lysed but only 70% of isolate NCKH_B and 60% of isolate NCKH_D were lysed in the first hour. During the course of the experiments, we discovered two populations of survival cells of NCKH_B and NCKH_D transformed into pseudocysts, but not of the ATCC strain. When the drug has been removed, the survival cells of NCKH_D resumed activity rapidly, but not those of NCKH_B. The CTC staining result showed NCKH_B pseudocysts have capacities of transforming to trophozoite after 3 hours of PHMB removal.
Also, we demonstrated that a double-drug combination efficaciously enhanced the anti-protozoan effect. In addition, to compare the invading capacities of NCKH_B and NCKH_D, we established a novel platform for ex-vivo NCKH infection. NCKH_B and NCKH_D cells are used to artificially infect cornea tissues. These results might aid to characterize the potential PHMB resistance mechanisms of Acanthamoeba and to develop a more effective treatment regimen for Acanthamoeba keratitis

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