棘阿米巴是一種自由營生的寄生蟲，常存在水中，而土壤中也有發現其存在。當人類接觸到含有致病性棘阿米巴的水體，棘阿米巴原蟲可能會經由角膜損傷處侵犯角膜，造成伺機性的感染，造成棘阿米巴角膜炎。但致病過程中，棘阿米巴是如何破壞細胞引起損傷症狀，目前尚未釐清。本研究首先以固定影像曠時攝影的方式觀察棘阿米巴致病性蟲株 NCKH_D與細胞共同培養的過程，發現細胞在未與棘阿米巴接觸的情況下，仍發生皺縮與脫落。接著我們再透過細胞毒力測試與固定影像曠時攝影證實，僅僅加入NCKH_D的分泌性蛋白質與細胞進行共培養同樣會對細胞造成破壞。這些結果顯示，棘阿米巴會透過非接觸性的方式對細胞造成破壞。根據本實驗室先前對於棘阿米巴的分泌性蛋白質的蛋白質體分析中發現，胺肽酶M20/M25/M40僅分泌自致病蟲株，因此我們認為胺肽酶M20/M25/M40是棘阿米巴對細胞造成破壞的重要關鍵。我們將NCKH_D蟲株的胺肽酶M20/M20/M40進行選殖、大量表現並加以純化，也以此蛋白質製造出具專一性的多株抗體。在分別加入胺肽酶抑制劑與胺肽酶M20/M25/M40多株抗體和NCKH_D分泌性蛋白質反應後，發現可減輕棘阿米巴分泌性蛋白質對細胞造成的破壞。此外，這些受損脫落的細胞在回收清洗後重新培養，發現仍然可以順利重新貼附再次生長。所以我們認為棘阿米巴的分泌性蛋白質會造成細胞脫落，但不是直接殺死細胞。另一方面，我們發現棘阿米巴胺肽酶M20/M25/M40抗體具有高度開發潛力，未來有望發展成為可用於環境水體與檢體樣本的快速診斷套組。

In this study, we found that the pathogenic Acanthamoeba could secret Aminopeptidase M20/M25/M40 to disrupt the co-cultured cells. By adding Aminopeptidase inhibitor and Aminopeptidase M20/M25/M40 specific polyclonal antibodies can reduce the situation. These results show that Aminopeptidase M20/M25/M40 is one of the virulence factors in Acanthamoeba. In addition, we also revealed the potential of the specific polycolonal antibody to develop the diagnosis kit.

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