臺灣博碩士論文加值系統

登革病毒(Dengue virus, DENV)已成為一個全球性的健康議題，因目前仍無有效的疫苗或抗病毒藥物可供臨床應用。所以針對登革熱的治療和預防方法是一個迫切的議題。海洋天然物為一新興藥物開發的領域，本研究旨在評估由海洋軟珊瑚中所萃取的天然物是否能抑制登革病毒的感染。初步篩選12種化合物，其中由海洋指形軟珊瑚中命名為WS 9-5、WS 9-7之萜類化合物具有最佳抗登革病毒感染的效果，WS 9-5對BHK細胞毒性CC50濃度為21.3 ± 6.4 μM，WS 9-7的CC50為47.6 ± 8.8 μM。以免疫螢光染色法評估WS 9-5抑制登革病毒非結構蛋白質1(Non-structure protein 1, NS1)的表現，顯示其抑制NS1蛋白表現效果EC50約為8.2 μM。同樣以免疫螢光染色法觀察，WS 9-5也顯示出有抑制登革病毒雙股RNA(dsRNA)生成的效果其EC50約為6.8 μM。除此之外，WS 9-5及WS 9-7也會影響登革病毒感染細胞後之病毒顆粒的產生。進一步利用DENV次基因體複製子(DENV replicon)方式檢驗WS 9-5，WS 9-7有無抑制病毒蛋白轉譯及病毒基因體的複製，結果顯示WS 9-5、WS 9-7並無抑制DV replicon的表現。另一方面檢視WS 9-5，WS 9-7是否透過誘導細胞產生先天性的免疫分子機制進而抑制病毒的感染，以IFN-β(interferon beta)啟動子及NF-ҡB(Nuclear transcription factor)結合位啟動子之報導基因評估WS 9-5、WS 9-7的影響，結果顯示細胞處理WS 9-5、WS 9-7並無誘導IFN的產生及NF-ҡB的活化。若前處理細胞後再感染病毒，相較於化合物WS 9-7，化合物WS 9-5的前處理時間越長(~ 6 hr)越有明顯抑制登革病毒NS1的表現，且先處理化合物WS 9-5並不影響細胞與病毒的結合。若先將登革病毒混合化合物WS 9-5及WS 9-7後再感染細胞，化合物WS 9-5及WS 9-7能明顯的中和病毒結合到細胞，且對日本腦炎病毒也有相同的效果。綜合以上，由指形軟珊瑚中分離出之萜類化合物WS 9-5及WS 9-7之抑制登革病毒感染的機制，可能是能結合上登革病毒並具有中和的效果而阻止登革病毒的黏著(Attachment)、進入(Entry)及脫鞘(uncoating)而導致後期的轉譯(Translation)、複製(Replication)、釋放(Release)被抑制。這是海洋天然物研究中，第一個發現海洋天然物以此機轉抑制登革病毒的感染，可進一步研究發展成有效的抗登革病毒藥物。

Dengue virus (DENV), becomes one of the reemerging infectious pathogens worldwide. Currently there are still no effective vaccines or anti-viral drugs in clinical. Thus, current researches in developing vaccines and antiviral drugs against dengue infection would be an urgent issue. Marine natural products have been emerging as a new era in drug development, and this study focus on searching the natural compounds from soft coral and then evaluating their effects to inhibit dengue virus infection. Twelve marine compounds were initially screened and compounds named WS 9-5 and WS 9-7 are potent in inhibiting dengue virus infection. The cell cytotoxicity (CC50) of the two terpenoids was evaluated by MTT assay. WS 9-5 is evaluated to be around 21.3 ± 6.42 μM, and WS 9-7 is 47.6 ± 8.82 μM. Immunofluorescence staining evaluations have concluded that WS 9-5 inhibits DENV protein expression (Non-structure protein 1, NS1) with an effective concentration (EC50) of approximately 8.3 μM and its double-stranded RNA (dsRNA) expression with EC50 around 6.8 μM. In addition, compound WS9-5 and WS9-7 were also able to show their impact on DENV virus particle production in which WS 9-5 shows better inhibition rate as compared to WS 9-7. Further, WS 9-5 and WS 9-7 had no inhibitory effect on DENV replication and translation as the luciferase activity of DENV sub-genomic replicon (DENV replicon) has not been suppressed. It is well known that upregulation of innate immune molecules, such as IFN-β and NF-κB, could show an immense down-regulation of DENV infection. However, the inhibitory effects exerted by WS 9-5 and WS 9-7 did not involve the innate antiviral activities as type I interferon-β and NF-κB were not activated in promoter reporter assay. Unexpectedly, premixing WS 9-5 and WS9-7 with In order to know the inhibitory mechanism mediated by WS 9-5 and WS 9-7, pretreatment of cells with compounds 1 hr before infection show no inhibitory effect, but longer incubation for 3 or 6 hrs before infection substantially inhibits virus infection. These results indicate WS 9-5 and WS 9-7 may interfere with the early steps in virus infection. However, compounds will not affect the expression of DV receptors on host cells. Unexpectedly, premixing WS 9-5 and WS 9-7 with virus not only effectively neutralizes virus binding to Huh7 and BHK cells but also suppresses virus infection. This neutralizing activity of WS 9-5 and WS 9-7 was also demonstrated in Japanese Encephalitis Virus infection. Collectively, results suggest that the marine compounds WS 9-5 and WS 9-7 are capable of inhibiting the dengue virus infection through the reduction in attachment, entry, and uncoating, thereby affecting the following translation and replication. Hence this data demonstrate that WS 9-5 WS 9-7 are contemplated to be the effective drugs with high potency in inhibiting the dengue virus infection.

摘要 II
Abstract IV
目錄 VI
一、 緒論 1
1.1 登革熱背景介紹 1
1.2 登革熱致病原 1
1.3 登革病毒各蛋白的功能 2
1.4 登革病毒感染及臨床表現 3
1.5 登革熱治療 4
1.6 抗登革化合物篩選 4
1.7 海洋化合物介紹 5
二、 研究目的 7
三、 實驗方法與材料 9
3.1 細胞培養 9
3.2 軟珊瑚樣品的取得 9
3.3 登革病毒的培養 10
3.4 登革病毒斑的測定 10
3.5 細胞毒性試驗 11
3.6 免疫螢光染色 11
3.7 細胞轉染 12
3.8 DENV次基因體複製子 13
3.9 病毒結合試驗 13
3.10 統計分析 14
四、 結果 15
4.1 天然化合物之抗病毒篩選 15
4.2 WS 9-5和WS 9-7之細胞毒性試驗 15
4.3 WS 9-5和WS 9-7之抑制登革病毒NS1抗原的表現 15
4.4 WS 9-5和WS 9-7之抑制病毒dsRNA的表達 16
4.5 WS 9-5和WS 9-7無法抑制DV replicon的表現 16
4.6 IFN-β及NF-ҝB的表現不參與WS 9-5和WS 9-7抑制病毒的感染 16
4.7 WS 9-5和WS 9-7在登革病毒感染前及後處理皆有抑制登革病毒NS1抗原的表現 17
4.8 WS 9-5和WS 9-7前處理及混和登革病毒後感染細胞皆有抑制登革病毒Binding的作用 18
4.9 WS 9-5和WS 9-7在混和日本腦炎病毒後感染細胞有抑制日本腦炎病毒Binding的作用 18
五、 討論 21
5.1 目前抗登革病毒藥物發展 21
5.2 抗病毒機制 22
5.3 抗病毒篩選平台 23
5.4 未來應用 24
六、 參考文獻 25
七、 圖表 31
八、 附錄一 49

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