- 學位論文
以核糖核酸干擾抑制登革病毒感染人類DC-SIGN轉殖細胞
Inhibition of Dengue Viral Infection of Human-DC-SIGN-transfected Cells by RANi
摘要
登革熱是全球熱帶與亞熱帶的重要流行疾病，許多國家正同世界衛生組織積極防治疫情發展。為了研究登革病毒，許多研究學者都致力於發展合適的動物模式作為研究基礎，但直到如今仍缺乏與在人體感染過程中相仿的動物模式。然而我們已經知道當人體遭感染登革病毒時，病毒是經由DC-SIGN分子進入樹突細胞，在伍安怡老師實驗室一個有趣的研究中已發現，將原本不表現人類DC-SIGN的小鼠B淋巴瘤M12細胞株，使它穩定表現人類DC-SIGN之後，便可於感染登革病毒48小時內偵測到細胞內病毒核糖核酸和蛋白的量上升，且培養液中的病毒顆粒數也顯著升高。這證明即使在小鼠細胞內，只要因著表現人類DC-SIGN，便使得登革病毒能感染、進入並在該細胞內複製。為了更近一步加強此一研究的結論，我們利用核糖核酸干擾的技術，降低原本穩定表現於M12細胞上的人類DC-SIGN，希望能證明因為人類DC-SIGN表現量減少，使得登革病毒無法感染此類M12細胞，進而說明人類DC-SIGN在小鼠模式發展上的重要性與可能性。實驗結果發現編號第90號的核糖核酸干擾序列最為有效，可減低人類DC-SIGN表現至20%。令人驚訝的是，之前已被報導過同樣可降低人類DC-SIGN表現至20%的另一段序列，在此實驗中測試後，竟然是無效的。當人類DC-SIGN因帶著有效的核糖核酸干擾而下降時，此M12細胞接著以登革病毒感染，48小時內無法表現出病毒蛋白，顯示小鼠細胞原本因為人類DC-SIGN的表現而獲得的被感染能力，再度因著核糖核酸干擾作用而失去受登革病毒感染的能力，也顯出DC-SIGN對於幫助非人類細胞獲得登革病毒感染的重要。
並列摘要
Dengue fever is an important epidemic disease distributing in tropic and sub tropic region. Many countries and WHO work hard to prevent its development. To investigate dengue virus, many researchers have devoted themselves to finding suitable animal model. Until now no animal model is comparable with human case. Dengue virus is infected in human body by interacting with DC-SIGN molecule on dendritic cells. There was an interesting result from previous study at Dr. Wu’s lab. Human DC-SIGN（hDC-SIGN）was stably expressed in the murine B lymphoma cell line M12 which cannot be infected by dengue virus originally. They found those M12 cells carrying hDC-SIGN became susceptible for dengue virus. Both dengue viral RNA and protein level increased within 48 hour post-infection. The number of dengue viral particle in supernatant also arose greatly. This result suggests that the expression hDC-SIGN is sufficient for dengue virus infection, entry and replication in murine cells. To further confirm this point, we design an RNA interference（RNAi）approach to knock down hDC-SIGN expression. By decreasing the expression of hDC-SIGN in M12 cells, dengue virus can not infect those cells anymore. The result may support the importance and the possibility of hDC-SIGN on establishing the murine model for dengue viral study. In this study we find No. 90 RNAi sequence is most efficient to reduce 80% of surface hDC-SIGN expression. Surprisingly, an RNAi sequence which can also knock down 80% of hDC-SIGN by published report previously, doesn’t work well in this experiment. Dengue viral protein could not be detected in hDC-SIGN-overexpressed M12 cells within 48 hours post-infection because of the decreased level of hDC-SIGN by RNAi（No. 90）. These data show that murine B lymphoma cell line M12 could gain the ability of susceptible for dengure virus with hDC-SIGN on its cell surface. On the other hand, level of hDC-SIGN could be down-regulated by RNAi and thus M12 cells cannot be infected by dengue virus. These findings conclude the importance of hDC-SIGN to make non-human cell become suitable for dengue viral infection.
參考文獻
延伸閱讀
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