臺灣博碩士論文加值系統

流行性感冒病毒屬於正黏液病毒科(Orthomyxoviridae)，每年在流感季都造成約300萬至500萬件重病案例，其中有約25萬至50萬名患者死亡。現今能實際用以治療流感病毒感染的藥物僅有NA神經氨酸酶的特異性抑制劑包括oseltamivir ( 克流感, Tamiflu ) , zanamivir及peramivir以及RdRp抑制劑favipiravir ( Avigan )。但流感病毒RdRp無校正功能，病毒在複製時易產生突變導致流感病毒易產生抗藥性，且造成疫苗保護效果有限，所以我們希望以老藥新用的方式，找出新的抑制流感病毒的藥物。此外，如同其他病毒，流感病毒必須仰賴宿主細胞的細胞因子 ( cell factor ) 完成病毒生活史，若篩選出來的藥物作用於細胞因子，則較能降低流感病毒突變所產生的抗藥性。
　　本實驗室之前利用RdRp報導系統已篩選出的六種可能抑制流感病毒的藥物，此六種藥物皆為上市的藥物，在安全上有一定的保障，我們試圖將這六種可能抑制流感病毒的藥物進行組合，期能產生協合作用 ( synergistic effect )，在低的使用劑量，亦能有效抑制病毒生長，如此也能降低病毒產生抗藥性的機率。
　　首先測試這六種藥物抑制流感病毒感染的能力。我們先是定義出六種藥物的CC50和IC50，發現其中有四種藥物能夠抑制流感病毒感染。緊接著將這六種藥進行二個藥物組合及三個藥物組合發現特定二配方組合為最佳藥物組合，有明顯的協同作用，能大幅抑制流感病毒感染。利用不同的加藥時間點，我們初步證明此二組合作用在病毒感染早期；利用血球凝集試驗，我們排除藥物作用於HA蛋白與受體結合。目前的實驗結果支持此藥物組合作用於RNP進入細胞核前的感染步驟。未來可利用動物模式進一步測試其抗病毒的效力。

英文摘要
Influenza virus belongs to Orthomyxoviridae. Influenza spreads around the world yearly, resulting in about three to five million cases of severe illness and about 250,000 to 500,000 deaths. The only drugs currently available to treat influenza are neuraminidase ( NA ) inhibitors,including oseltamivir ( Tamiflu ), zanamivir and peramivir, and an RNA-dependent RNA polymerase ( RdRp ) inhibitor, favipiravir ( Avigan ). Since influenza virus RdRp lacks proofreading activity, the genome is easy to mutate when replicating. As a result, drug resistance and limited vaccine protection occur. In this project, we hope to repurpose FDA-proved drugs for treating influenza. As to all viruses, influenza virus depends heavily on cellular mechanism to complete its life cycle. If inhibited cellular factors mediate the antiviral effects ( cellular factor-targeting ), drug-resistant influenza viruses may not be selected easily by cellular factor-targeting drugs as compared to virus-targeting drugs.
Previously our lab used RdRp reporter assay to screen drugs, and found six drugs may inhibit influenza virus infection. All six drugs are generic drugs. Here I looked for combinations with synergistic anti-viral effects even at low drug dosages. A lower drug dosage will assert lower selection pressure on influenza virus and less cytotoxicity on host. In addition, treatment with multiple drugs decrease the possibility of drug-resistant viruses.
First, the CC50 and IC50 of each of these six drugs were determind on MDCK and influenza-infected MDCK cells, respectively. Therapeutic index for each compound was calculated. Four out of six drugs tested were found to inhibit influenza virus replication by itself. Next, all of the binary and ternary combinations were tested, which concluded that a particular binary combination is most effective. In order to identify the likely steps suppressed by this combination, I added the drug combination at different time points post infection in time course assays and found that combination inhibited at early stage of the infection cycle. The results of hemagglutinin assay excluded that the combination inhibited HA protein binding to its receptor. Instead, the preliminary results supported that the combination acts on the step prior to viral RNP into cell nucleus. Testing the anti-viral ability of the combination on an animal model will further demonstrate its efficacy and potential in the future.

誌謝 i
中文摘要 ii
英文摘要 iv
目錄 v
圖目錄 vii
表目錄 viii
第一章 緒論 1
第一節 流行性感冒病毒簡介 1
第二節 現存抗流感病毒藥物 4
第三節 RdRp報導系統 ( reporter system ) 7
第四節 六種藥物簡介 9
第五節 藥物有效濃度計算軟體 ( compusyn ) 15
第六節 實驗目的 16
第二章 實驗材料與方法 18
第一節 實驗材料 18
第二節 實驗方法 22
第三章　結果 32
一. 六種藥物不同濃度與降低RdRp報導能力呈現正相關 32
二. 六種藥物毒性測試 33
三. 六種藥物對抑制PR8及pdm 2009流感病毒感染效果測試 34
四. 尋找具有協同作用的藥物組合 36
五. Auranofin + pimozide作用在病毒感染的早期 38
六. Auranofin + pimozide無法直接與流感病毒HA蛋白結合 40
七. Auranofin + pimozide抑制RNP進入細胞核內 40
八. Auranofin或pimozide單獨使用降低病毒量 42
九. Auranofin或pimozide單獨使用作用在病毒感染的早期 42
十一. Auranofin或pimozide皆可以抑制RNP進入細胞核內 44
第四章　結論 46
第一節 個別藥物抑制流感病毒感染 46
第二節 藥物組合抑制流感病毒感染 48
第五章　討論 50
第六章　圖 53
第七章　表 84
參考文獻 87
附錄 96

圖次
圖1. 不同濃度的六種藥物降低RdRp報導能力 53
圖2. Tamiflu和六種藥物細胞毒性與藥物濃度關係 55
圖3. 藥物對抑制PR8流感病毒感染效果 57
圖4. 藥物對抑制pdm 2009流感病毒感染效果 59
圖5. 2種藥物組合或3種藥物組合對抑制PR8流感病毒感染效果 61
圖6. 2種藥物組合或3種藥物組合對抑制pdm 2009流感病毒感染效果 63
圖7. Auranofin + pimozide降低病毒viral titer 65
圖8. Auranofin + pimozide作用在病毒感染的早期 67
圖9. Auranofin+ pimozide無法直接與PR8或pdm 2009流感病毒HA蛋白結合 70
圖10. Auranofin + pimozide抑制RNP進入細胞核內 72
圖11. Auranofin或pimozide單獨使用降低viral titer 75
圖12. Auranofin或pimozide單獨使用作用在病毒感染的早期 77
圖13. Auranofin或pimozide無法與流感病毒HA protein結合 79
圖14. Auranofin或pimozide皆可以抑制RNP進入細胞核內 81

表次
表1. 統整七種藥物藥物毒性與效果 84
表2. 兩種藥物組合感染病毒後細胞存活率 85
表3. 三種藥物組合感染病毒後細胞存活率 86

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