登革熱病毒藉由人類DC-SIGN感染小鼠B淋巴球

Human DC-SIGN mediates dengue virus infection of mouse B lymphocytes

林大鈞(Ta-Chun Lin)

指導教授 ： 伍安怡

國立臺灣大學/醫學院/免疫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02938

摘要

登革熱病毒是一種由蚊子傳染的疾病，近年來登革熱已成為世界性的公衛問題，可是因缺乏好的動物模式，其致病及出血機制仍不清楚。因此，建立一個動物模式來進行這方面研究就成為不可或缺的前提。
人類DC-SIGN受體主要表現在人類樹突狀細胞上，已被報導可以辨認登革熱病毒並且幫助病毒進入細胞內。因此我假設小鼠細胞表現人類DC-SIGN受體便可以幫助登革熱病毒進入小鼠細胞內，進而造成病毒在小鼠細胞內繁殖。長遠來看，若假設成立，那麼我們便可以建立一隻轉殖基因小鼠表現此受體作為研究登革熱病毒感染致病機制的動物模式。
首先，我比較人類Raji細胞株與人類Raji細胞株表現人類DC-SIGN對於感染登革熱病毒所造成的差異。我發現當人類Raji細胞株在表現人類DC-SIGN後，可以成為登革熱病毒的宿主細胞。接著我建立一穩定表現人類DC-SIGN的小鼠B細胞株，藉由比較有表現人類DC-SIGN與不表現人類DC-SIGN的小鼠B細胞株在感染登革熱病毒後的結果，我發現有表現人類DC-SIGN的細胞株，可以在細胞中表現病毒的core基因及13kDa的病毒蛋白。同時自感染後12小時開始，在感染細胞株的培養液中，病毒core基因的數量持續上升。這些結果證明了登革熱病毒可以藉由人類DC-SIGN進入小鼠細胞，同時也可在細胞中進行繁殖。另一方面，我也觀察表現人類DC-SIGN的小鼠B細胞在感染登革熱病毒後的細胞反應。發現細胞在被登革熱病毒感染後，表現CD69、CD86和分泌TNF-α及IL-6蛋白質都有升高的現象。這些結果暗示登革熱病毒不但可以在有表現人類DC-SIGN的小鼠細胞內繁殖，同時也可以引發細胞反應。
此外我設計了一個載體 (pK14Tyr-PolIIhDC-SIGN-IN2B)，使得人類DC-SIGN基因可以受小鼠第二聚和脢促進子的驅動，以及被兩份隔離子所保護。在轉殖此載體進入人類293T細胞後，人類DC-SIGN受體可表現在細胞表面。接著此載體以顯微注射遞送到小鼠受精卵中，其子代以聚合脢鏈鎖反應篩選轉殖基因是否嵌入其基因體。我發現18隻子代中有兩隻小鼠之基因體有表現人類DC-SIGN基因。接著我以其中的一隻小鼠周邊血白血球做蛋白質表現分析，也發現人類DCSIGN受體有表現在70%細胞表面，這個結果證明了此載體的可用性。以後利用這一隻轉殖基因小鼠來研究登革熱病毒感染的致病機制是可期待的。

關鍵字

登革熱病毒

並列摘要

Dengue virus is an arthropod-borne flavivirus that causes dengue fever and heamorrhagic fever in humans. It is an emerging and volatile public health concern, but the pathogenic mechanism has not been elucidated. An animal model is needed to study the pathogenesis of dengue virus infection.
It has been reported that human C-type lectin DC-SIGN that is expressed on human monocyte-derived dendritic cells binds to dengue virus. Thus, it is my speculation that animals expressing hDC-SIGN will facilitate dengue virus entering target cells and allow virus replication. The long term goal of our laboratory is to generate transgenic mouse lines to study the immunopathogenic mechanism of dengue. As a first step to reach that goal, the first aim of my study was to investigate whether mouse cells expressing hDC-SIGN became more susceptible to dengue virus infection. Next, I designed and constructed a plasmid to generate hDC-SIGN transgenic mice for the study of dengue immunopathogenesis.
First, I compared human B cell lines Raji and transfected Raji-DC-SIGN for their susceptibility to dengue virus infection and found that Raji-DC-SIGN cells were susceptible to dengue virus infection, verifying results reported in the literature. Later, I transfeced mouse B cell line M12 with hDC-SIGN and selected three stable clones, all with >90% of hDC-SIGN expression. Comparing hDC-SIGN expressing M12 with untrasnfected parental M12, I found that after infection dengue virus core gene mRNA and 13 kDa viral proteins were detected in hDC-SIGN-M12 but not in parental M12 at 12h. The virus copy numbers in the supernatants of hDC-SIGN-M12 culture also gradually increased at 12 h on after infection, indicating dengue virus replicates in the hDC-SIGN-transfected M12 cells. These results demonstrate that hDC-SIGN mediates dengue virus entry into mouse B cells and transfected mouse B cells support dengue virus replication. Furthermore, cellular response of hDC-SIGN-M12 to dengue virus was monitored. The results showed that infection by dengue virus upregulated the expressions of CD69 and CD86 and the production of TNF-α and IL-6 in hDC-SIGN-M12 cells. The results of these in vitro studies indicate that mouse cells can support the replication of and mount cellular responses to dengue virus, providing that hDC-SIGN is expressed.
I went on to construct a plasmid (pK14Tyr-PolIIhDC-SIGN-IN2B) that hDC-SIGN is under the control of polymerase II promoter, followed by a polyA tail and two copies of insulators. In vitro study showed that 293T cells transfected with pK14Tyr-PolIIhDC-SIGN-IN2B plasmid expressed hDC-SIGN on the surface. Offspring of the fertilized eggs whose pronuclei were injected of the plasmid were screened for transgene expression. Screening of the genomic DNA by PCR, I found two transgenic founders carried the hDC-SIGN transgene and the peripheral blood leukocytes of one of them expressed hDC-SIGN on the cell surface. These results showed that the strategy of plasmid construction works. The transgenic progeny will be ready for the study of the immunopathogenic mechanisms of dengue in the near future.

並列關鍵字

dengue virus ； DC-SIGN

參考文獻

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