1998年在台灣暴發腸病毒的大流行，最主要是由腸病毒71型引起，除了手足口病徵之外，有些病例中會引起腦炎、無菌性髓膜炎而造成流行性神經麻痺後遺症，嚴重者會導致死亡，其中嬰兒和小孩為最主要的感染者。有鑑於目前尚無有效疫苗抑制腸病毒71型的感染，因此致力於發展腸病毒疫苗已成為重要目標。RNAi干擾現象是由21-nt siRNAs引起基因沉默的現象，近年來利用RNAi抑制基因表現的機制已在哺乳動物細胞內被廣泛研究。目前已有許多研究指出RNAi可用於哺乳動物細胞對抗病毒的感染上。因此本實驗根據RNAi機制特性，使用長片段dsRNA應用於抑制腸病毒71型的複製。首先利用pol II啟動子轉錄長片段dsRNA，讓RNAi抑制效用比較穩定，因此建構兩個含不同pol II啟動子的載體，分別為(1) pTCY載體：帶有β-actin啟動子可持續於細胞內表現基因，(2) p2-5A載體：含有2'-5'(A)n酵素合成酶基因啟動子，其受干擾素調控基因表現的能力。當腸病毒71型感染細胞時，病毒表現2A蛋白質切酶為切割病毒蛋白之重要切酶，因此選擇2A基因做為本實驗之標的基因。接著，將大小約為450 bp之2A基因片段以正反接方式接入pTCY和p2-5A載體，命名其為pTCY-ds2A及p2-5A-ds2A。將構築好的兩載體分別轉染Vero細胞後，以腸病毒液感染轉染ds2A的Vero細胞，得到抑制病毒能力較高的細胞株Vero/pTCY-ds2A-2和Vero/p2-5A-ds2A-2。根據細胞病變現象分析，轉染ds2A的細胞株抑制病毒能力高於正常細胞1000倍，且可減少細胞死亡現象。進一步利用RT-PCR檢測腸病毒71型mRNA是否因RNAi作用而減少，以3D基因作為檢測標準，此基因會轉譯為聚合酶，可使腸病毒71型進行複製；根據結果顯示Vero/pTCY-ds2A-2和Vero/p2-5A-ds2A-2兩細胞株皆減少腸病毒mRNA的複製。另分析細胞凋亡情形，發現轉染ds2A的兩細胞株可減少細胞DNA斷裂。經觀察RNAi效用的持續性結果顯示，Vero/pTCY-ds2A-2和Vero/p2-5A-ds2A-2細胞株皆可維持RNAi抑制能力11~14天；初步以西方轉漬法分析PKR蛋白質表現，在無病毒感染細胞的情況之下，轉染ds2A的兩細胞株內，PKR蛋白質表現量低而無明顯差別，顯示dsRNA的存在可能不誘導受干擾素調控基因的表現。為了證實細胞抗病毒現象確實與RNAi有關，利用Vero/pTCY-ds2A-2、Vero/p2-5A-ds2A-2和Vero/pTCY-dsE1A進行腸病毒感染實驗，觀察結果Vero/pTCY-dsE1A細胞株大部分細胞死亡和無轉染之Vero細胞並無差異，表示細胞病變具有專一性。綜合以上實驗，利用病毒2A 蛋白切酶基因長片段dsRNA而引起的基因沉默現象，可有效用於抑制腸病毒71型複製。

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