臺灣博碩士論文加值系統

兹卡病毒(Zika virus) 為蚊子媒介的黃病毒，近年爆發大規模感染，期間感染孕婦造成胎兒或新生兒罹患小腦症之比例顯著上升。目前茲卡病毒沒有疫苗預防或特效藥治療，開發抗茲卡病毒藥物為當前重要課題。AXL (受器酪胺酸激酶)為茲卡病毒共同受器之一，為開發抗茲卡病毒藥物標的之一。本論文將探討酪胺酸激酶抑制劑-馬來酸舒尼替尼(sunitinib malate)抗茲卡病毒之功效及其作用機制。首先，同時給藥試驗發現馬來酸舒尼替尼明顯濃度依賴地抑制茲卡病毒對BHK-21細胞病變。由百分之五十組織培養感染劑量試驗計算馬來酸舒尼替尼抑制感染細胞的病毒產率之百分之五十濃度(IC50)為0.0146 μM。於茲卡病毒感染 1及4小時後，再處理馬來酸舒尼替尼試驗發現其對病毒產率之IC50分別為 0.009 μM及0.052μM。Rear time PCR試驗顯示馬來酸舒尼替尼無影響感染三天後細胞內正股病毒RNA的合成，但是大量負股病毒RNA累積。另外，藉由免疫螢光分析觀察感染1或4小時候後再給藥試驗，茲卡病毒E蛋白表現程度，發現馬來酸舒尼替尼抑制E蛋白表現之IC50分別為0.202 μM及0.898 μM。在細胞內病毒顆粒試驗也發現馬來酸舒尼替尼造成細胞內有大量病毒顆粒累積，無法釋出。因此，推斷馬來酸舒尼替尼具有抑制茲卡病毒之晚期複製功效。

Zika virus is a mosquito-borne flavivirus, causing the increasing risk of the microcephaly and the other birth defect in fetuses and infants during the large outbreaks in recent years. Vaccines and antiviral agents are not yet available; developing anti-Zika virus drugs becomes an important issue on global health nowadays. AXL, a cell surface receptor tyrosine kinase, has been identified as Zika virus co-receptor, as an attractive target for developing anti-Zika virus agent. The study investigates the antiviral activity and related mechanism of a multitargeted tyrosine kinase inhibitor sunitinib malate against Zika virus. Initially, sunitinib malate significantly inhibited Zika virus-induced cytopathic effect, reducing Zika virus yield in BHK-21 cells. The TCID50 assay indicated that the IC50 value of sunitinib malate was 0.0146 μM for the virus yield reduction. Time-of-addition assay indicated that the IC50 values of sunitinib malate treatment 1 and 4 hr post-infection were 0.009 μM and 0.052 μM for the virus yield reduction, respectively. Real-time RT-PCR assay showed that sunitinib malate treatment had no effect on the synthesis of positive-strand viral RNA genomes, but prompted a large accumulation of negative-strand viral RNA genomes in infected cells. In addition, immunofluorescent staining indicated that IC50 values of sunitinib malate treatment 1 and 4 hr post-infection were 0.202 μM and 0.898 μM for the decrease of viral E protein, respectively. Intracellular virion particle assay demonstrated that sunitinib malate predominantly accumulated the large amount of infectious Zika virus particles in cytoplasm. The results revealed that a multitargeted tyrosine kinase inhibitor sunitinib malate has anti-Zika virus activity via blocking the late stage of viral replication.

摘要 I
Abstract II
目錄 IV
圖目錄 VI
表目錄 VII
第一章 前言 8
第一節 研究背景 8
一、 黃病毒科 8
二、 茲卡病毒發現與流行病學 10
三、 茲卡病毒基因型與生命週期 10
四、 茲卡病毒臨床症狀 11
五、 茲卡病毒治療與疫苗 12
六、 馬來酸舒尼替尼 (sunitinib malate) 13
第二節 研究目的 14
一、 實驗動機 14
二、 實驗目的 14
第二章 研究方法 14
第一節 研究設計 14
一、 抗病毒活性試驗 15
二、 抗病毒機制之探討 15
第二節 研究材料 17
一、 使用之細胞株 17
二、 使用之病毒株 18
三、 抗體&引子 18
四、 實驗儀器及藥品 19
第三節 實驗方法 20
一、 細胞存活率試驗(MTT assay) 20
二、 細胞病變抑制實驗 21
三、 流式細胞儀檢測細胞週期染色 22
四、 半數組織培養感染劑量(TCID50) 23
五、 細胞半有效濃度試驗(IC50) 23
六、 免疫螢光染色分析(IFA) 24
七、 病毒顆粒活性抑制試驗(Virucidal) 25
八、 病毒顆粒貼附能力抑制試驗(Attachment) 25
九、 對病毒作用之時間點分析(Time of addition assay) 26
十、 酵素結合免疫吸附分析法（Enzyme-linked immunosorbent assay，ELISA) 26
第三章 研究結果 28
第一節 Sunitinib對BHK-21細胞之半數細胞毒殺劑量 29
第二節 茲卡病毒效價 30
第三節Sunitinib對茲卡病毒的抗病毒能力 30
第四節 Sunitinib抗茲卡病毒機制 32
第四章 討論 37
第五章 結論與建議 40
第一節 結論 40
第二節 建議 40
參考文獻 68

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