臺灣博碩士論文加值系統

A型流行性感冒病毒是季節性盛行的病毒，對於人類與動物具有高度傳染與感染率，導致有高度的致死率。雖然目前有抑制流感病毒的藥物，但病毒的不斷演化，開始對藥物產生了抗藥性，因此，開發新的抗流感病毒藥物是需要的工作。本研究旨在探討奧氏虎皮楠抑制流感病毒的功效。首先，以細胞存活試驗，篩選出不具細胞毒殺性的奧氏虎皮楠分離化合物；接著，再利用病毒斑抑制試驗觀察化合物對流感病毒A/WSN/1933(H1N1)的抑制效果。其中化合物(DGNA-4N15)對流感病毒H1N1的50%有效抑制病毒濃度為2.316 ± 0.80 μM；此化合物也對流感病毒H3N2有抑制效果，顯示此化合物具有廣泛抑制流感病毒的潛力。接著，以病毒斑抑制試驗及免疫螢光試驗分析藥物抑制在病毒感染的哪個階段；實驗證實DGNA-4N15可有效抑制流感病毒H1N1進入細胞的早期階段。接下來，我們以紅血球凝集抑制試驗觀察化合物阻斷流感血凝素與唾液酸受體的交互作用，確定DGNA-4N15可以阻斷流感血凝素與唾液酸受體的交互作用，綜合上述，本研究發現奧氏虎皮楠化合物DGNA-4N15能有效阻止流感病毒進入宿主細胞，有機會發展為抗流感病毒的先導型化合物。

Influenza A virus is, a seasonal prevalent virus which is highly contagious and infectious to humans or animals and may lead to high mortality. Though there are therapeutic drugs , the continuous evolution makes the drug-resistant strains occur. Therefore, the development of new anti-influenza virus agents is needed. In this study, we found that Oldham Daphniphyllum can inhibit influenza virus infection. Firstly, the cell cytotoxicity assay was used to screen the non-toxic compounds derived from Oldham Daphniphyllum and then the anti-influenza A virus activity was determined by plaque assay. A compound, DGNA-4N15, exhibited a highly anti-viral activity. The concentration of 50% inhibition effect (EC50) of DGNA-4N15 against influenza virus A/WSN/1933(H1N1) is 2.316 ± 0.80 μM. The compound also inhibits A/Brisbane/10 /2007 (H3N2) virus ( EC50= 5.27 ± 2.142 μM ). It implied that the compound may broadly inhibit influenza viruses. Following, the different stages of viral infection inhibited by the compound were analyzed by plaque assay and immunofluorescence assay. The compound was found to effectively inhibit the entry of influenza A virus (H1N1) into cells. Moreover, we observed that DGNA-4N15 can block the interaction between influenza hemagglutinin and cell sialic acid receptors by hemagglutination inhibition assay. Above all, we demonstrate that the compound DGNA-4N15 derived from Oldham Daphniphyllum can effectively inhibit influenza A virus in the early step of virus infection and may have an opportunity to develop as a lead compound to against influenza virus.

目錄
中文摘要 I
英文摘要 II
目錄 III
圖目錄 VI
表目錄 VII
縮寫表 1
第一章 前言 2
第一節 研究背景 2
1-1. 流行性感冒病毒的介紹 2
1-2 流感病毒的特性 3
第二節 A型流感生活史 4
第三節 流感病毒的預防疫苗與抗流感藥劑治療 6
第四節 奧氏虎皮楠的簡介 8
研究方向 8
第二章 材料與方法 10
第一節 實驗設計 10
第二節 研究材料 10
1. 化合物來源 10
2. 化學藥品 10
第三節 實驗方法 12
1. 細胞培養 12
2. 細胞存活率試驗 12
3. 病毒製備 12
4. 病毒斑分析(plaque assay) 13
5. 病毒斑減低分析(plaque reduction assay) 14
6. 免疫螢光分析試驗 (Immunofluorescence assay ; IFA) 14
7. 誘導病毒膜融合(Acid-bypass treatment) 15
8. 標定流感病毒(Influenza virus labeling) 15
9. 血球凝集試驗(Hemagglutination test ; HA test) 15
10. 流式細胞術(Flow cytometry) 16
11. 統計分析方法 16
第三章 結果 18
第一節 化合物篩選 18
第二節 探討DGNA-4N15對流感病毒不同時期影響 18
第三節 探討DGNA-4N15抑制病毒進入細胞核 20
第四節 探討DGNA-4N15與流感病毒的脫殼表現 20
第五節 探討DGNA-4N15抑制病毒進入宿主細胞 21
第六節 探討DGNA-4N15流感病毒與細胞的結合表現 21
第七節 探討DGNA-4N15是否可以抑制流感病毒HA的活性 23
第四章 討論 24
第五章 結論 27
參考文獻 29


 
圖目錄
圖一、篩選無細胞毒殺性的奧氏虎皮楠化合物 32
圖二、篩選抗流感病毒活性的奧氏虎皮楠化合物 33
圖三、觀察衍生物DGNA-4N15的抗病毒活性 34
圖四、化合物DGNA-4N15在不同時期抗病毒活性 35
圖五、化合物DGNA-4N15對病毒感染進入宿主細胞核之影響 36
圖六、探討化合物DGNA-4N15在膜融合策略中的影響 37
圖七、探討化合物DGNA-4N15在Mix-treatment策略中的影響 38
圖八、觀察化合物對病毒與宿主細胞Attachment時期的影響 39
圖九、觀察化合物DGNA-4N15影響病毒感染宿主細胞 40
圖十、觀察化合物DGNA-4N15影響病毒在細胞表面的表現 41
圖十一、觀察化合物DGNA-4N15對病毒在血球凝集試驗中的影響 42
 
表目錄
表一、觀察化合物DGNA-4N15加藥影響的結果 43
表二、觀察化合物DGNA-4N15在不同流感病毒株加藥影響的結果 44
表三、觀察化合物DGNA-4N15抑制病毒在血球凝集試驗中的結果 45

 

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