臺灣博碩士論文加值系統

腸病毒71型為小RNA病毒科中腸病毒屬的ㄧ員，主要是感染幼童，而多數患者的症狀為較輕微的手足口症，然而少數的嚴重感染則會導致神經方面的併發症。目前並無臨床上所使用的抗腸病毒藥物，而腸病毒藥物的研發也持續在進行，其策略包含針對病毒本身的設計，或是透過藥物篩選的方式，另外也可藉由小兒麻痺病毒及鼻病毒相關藥物之改良與應用；然而此類抗病毒藥物的發展屬針對病毒本身進行抑制，因此具產生抗藥性病毒的可能性。病毒感染時，會誘發病毒複製所需之宿主基因表現或是相關的訊號傳遞路徑被活化，因此我們使用LOPAC化合藥庫進行抗腸病毒藥物的篩選，而LOPAC化合藥庫含1280種已知生物活性及功能的小分子化合物，藉以篩選出會抑制病毒複製所需的基因或訊號傳遞路徑之藥物。透過先前實驗室利用螢光共振能量轉移原理所建立的生物感測平台，此細胞株可穩定表現一質體，帶有綠色螢光蛋白GFP2，以及紅色螢光蛋白DsRed2，中間的多肽連結為2A蛋白酶之切點，當腸病毒71型感染時，藉螢光的偵測即可即時偵測並定量腸病毒71型於細胞中的感染情形。將此生物感測細胞株應用於高效能藥物篩選上，其Z’ factor為0.53，顯示此平台具穩定性及可信度，並篩選出15種藥物的FRET比值提升高於50%。其中，E化合物為用於治療阿米巴的臨床用藥，因此選擇此藥物進行更進一步的研究。E化合物的EC50與CC50分別是1.19 μM和大於4096 μM，經計算得知其選擇性指標，SI值高於3439.1。E化合物可減緩腸病毒71型感染所造成的細胞病變及死亡，且其抑制效率和藥物濃度也呈現正相關。經由在病毒複製週期的不同時間點加藥之試驗，得知E化合物作用於病毒感染後的0至5小時，並了解到E化合物會抑制病毒的蛋白質合成，以及病毒基因體的複製；但不會選擇性影響病毒的轉譯作用以及蛋白酶的切割能力。將E化合物創新應用於腸病毒的治療上，可提供一新的治療方式及策略以控制腸病毒71型的感染。

Enterovirus 71 (EV71), a member of the genus Enterovirus of the family Picornaviridae, is a common pathogen often associated with mild foot-and-mouth disease and occasionally severe neurological manifestations in young children. However, there have been no anti-EV71 drugs available for clinical use. Several strategies for anti-EV71 drug development have been employed, ranging from viral target-based design, compound screening, and compound series generated from antiviral developments against related poliovirus and human rhinoviruses. All these efforts exclusively focused on viral targets, which likely develop drug resistance. Viral infection can induce host genes and cellular signaling pathways that are essential for virus propagation. Therefore, we proceeded with a screen from the Library of Pharmacologically Active Compound (LOPAC; Sigma–Aldrich) that consist of 1280 small molecules with well-defined bioactivities against diverse cell functions for hits that could intervene the activated, virus-supporting cellular genes/pathways. Using fluorescence resonance energy transfer (FRET), our laboratory previously developed a genetically engineered, FRET-based biosensor achieved by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 and red fluorescent protein 2, with a cleavage motif of the EV71 2A protease connected in between. The HeLa cell based-FRET biosensor showed a real-time and quantifiable impairment of FRET proportional to EV71 replication. We first assessed the adaptability of the FRET-biosensor as a high-throughput drug screen platform, and showed its rapidity and robustness with a Z’ value at 0.53. Among fifteen compounds that significantly (>50%) elevated the FRET ratio, E compound, a clinical drug for treating amebiasis, was chosen for further study. The 50% effective concentration (EC50) and 50% cytotoxicity concentration (CC50) are 1.19 μM and >4096 μM, respectively, making the selectivity index >3439.1. E compound significantly protected the infected cells from the cytopathic effect and cell death caused by EV71 in a dose-dependent manner. From the time-of-addition experiment, we identified the E compound action time at about 0~5 h pi. We further showed that the compound effectively intervened the virus lifecycle at the stages of the viral protein synthesis, genome replication but not the viral translation initiation and protease activity. This new use of E compound may provide new perspectives for therapeutic strategies for controlling EV71 infections and their consequences.

目錄
目錄 i
中文摘要 iv
Abstract v
第一章 緒論 1
第一節 腸病毒71型的發現 1
第二節 腸病毒之分類及特性 1
第三節 腸病毒的基因體結構與蛋白質 2
第四節 病毒的複製週期 3
第五節 腸病毒71型的流行病學及臨床概況 4
第六節 臨床概況與抗病毒藥物及治療之發展 5
第七節 LOPAC化合物藥物庫之簡介與應用 8
第八節 穩定表現螢光共振能量轉移之細胞株 9
第九節 研究動機與目的 11
第二章 材料與方法 12
第一節 細胞株的培養與操作 12
第二節 病毒來源、培養與定量 15
第三節 LOPAC化合物藥庫之篩選 18
第四節 E化合物濃度和FRET Ratio間之相關性 20
第五節 E化合物和腸病毒71型感染所造成細胞死亡之間的關係 22
第六節 觀察E化合物對於病毒感染所造成之細胞病變的影響 24
第七節 E化合物對腸病毒71型抑制之EC50 26
第八節 E化合物之細胞毒性試驗及CC50之計算 28
第九節 利用免疫螢光探討不同時間點下處理E化合物對於腸病毒71型複
製週期之影響 30
第十節 探討E化合物對於轉染表現之腸病毒71型IRES活性之影響 33
第十一節 利用細胞外轉譯試驗鑑定腸病毒71型IRES和E化合物之交
互作用 36
第十二節 研究E化合物與腸病毒71型2A蛋白酶之切割能力間的關係
42
第十三節 研究E化合物與腸病毒71型3C蛋白酶之切割能力間的關係
48
第十四節 探討E化合物對腸病毒71型RNA的影響 52
第十五節 E化合物處理對病毒蛋白質合成的影響 57
第三章 實驗結果 62
第一節 自LOPAC化合藥物找尋有效抑制腸病毒71型之藥物 62
第二節 E化合物之有效抑制濃度及細胞毒性並評估其選擇性指數 64
第三節 E化合物對於腸病毒71型複製週期的影響 66
第四章 討論 70
第五章 圖表 73
圖一、篩選策略及藥物篩選平台的穩定性 75
圖二、LOPAC1280化合藥庫篩選結果及藥物分類 76
圖三、E化合物濃度與FRET ratio呈正相關 77
圖四、E化合物可抑制病毒感染所造成的細胞死亡 78
圖五、E化合物可減緩病毒所造成的細胞病變 79
圖六、根據選擇性指數的評估，E化合物使用的安全性高 80
圖七、免疫螢光試驗分析不同病毒複製時間點加藥之影響 81
圖八、E化合物與腸病毒71型IRES活性之相關性 82
圖九、E化合物對於2A蛋白酶的切割活性無抑制作用 83
圖十、3C蛋白酶之切割能力不會受到E化合物的影響 84
圖十一、病毒RNA受到E化合物的抑制 85
圖十二、E化合物之濃度與病毒蛋白表現量的抑制效果呈正相關 86
第六章 參考文獻 87
第七章 附錄 95
附錄一、微小核糖核酸病毒分類 95
附錄二、腸病毒71型病毒結構 96
附錄三、腸病毒71型基因體及其蛋白質 96
附錄四、腸病毒71型生活史 97
附錄五、腸病毒71型動力學於人類橫紋肌肉瘤細胞株 97
附錄六、台灣腸病毒流行現況 98
附錄七、2012-2013年台灣腸病毒重症疫情趨勢圖 98
附錄八、腸病毒的感染途徑 99
附錄九、抗腸病毒71型之藥物相關結構 99
附錄十、腸病毒71型抑制劑作用機制之概要 100
附錄十一、GFP2-DsRed2融合螢光蛋白經病毒感染後螢光變化之概念圖 101
附錄十二、GFP2與DsRed2激發及放射光譜 102
附錄十三、E化合物之化學結構 102

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