研究標靶細胞內吞小體酸化作用以抑制登革病毒感染

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Title:	研究標靶細胞內吞小體酸化作用以抑制登革病毒感染 Study on the Blockade of Dengue Virus Infection by Targeting Endosomal Acidification
Authors:	何敏如 Ho, Min-Ru
Keywords:	登革病毒;神經元細胞;神經毒殺作用;病毒性腦炎;內吞作用 Dengue virus;Neuronal cells;Neurotoxicity;Encephalitis;Endocytosis
Date:	2018
Issue Date:	2019-01-02 12:16:28 (UTC+8)
Abstract:	登革病毒感染神經元細胞被認為會引發神經病變。本研究中我們利用細胞及動物實驗模式，藉由標靶細胞內吞小體酸化作用，探討登革病毒感染神經元細胞的潛在途徑以及阻斷登革病毒感染的可能方式。實驗室先前的研究顯示，在登革病毒感染的小鼠腦中，於海馬迴區域之神經元細胞可測得病毒蛋白表現與神經細胞毒殺現象。登革病毒會造成小鼠神經元細胞株 (Neuro-2a) 的感染，包含病毒進入、病毒核糖核酸複製、病毒蛋白質表現、病毒顆粒釋放以及後續的細胞毒殺作用。小鼠神經元細胞株會表現多巴胺D2受體，而抑制多巴胺D2受體則可有效阻斷登革病毒感染。利用藥理學方式阻斷網格蛋白質媒介之內吞作用可抑制病毒複製。藉由標靶空泡型氫離子三磷酸腺苷酶媒介之細胞內吞酸化作用，可以有效地阻止登革病毒複製，但對病毒的轉譯作用沒有直接影響。此外，阻斷多巴胺D2受體、網格蛋白質和空泡型氫離子三磷酸腺苷酶媒介之細胞內吞酸化作用，亦可減弱登革病毒所誘導的神經毒殺作用。阻斷多巴胺D2受體和內吞酸化作用，可以在小鼠當中有效減緩登革病毒感染、急性病毒性腦炎和死亡率。這些結果表明，多巴胺D2受體和網格蛋白質媒介之內吞作用與後續細胞內吞酸化作用，幫助登革病毒感染進入神經細胞與複製，進而導致神經毒殺作用。 Dengue virus (DENV) infection in neuronal cells is speculated to trigger neuropathy. Here, we investigated the infectious route of DENV in neuronal cells and determined the blockade of DENV infection by targeting endocytic pathways in vitro and in vivo. In the DENV-infected brains, we previously showed viral proteins were expressed in neuronal cells around the hippocampus accompanied by neurotoxicity. DENV caused infection, including entry, dsRNA replication, protein expression, and virus release, followed by cytotoxicity in the mouse neuronal cell line Neuro-2a. Regarding dopamine receptor D2 (D2R) expression in Neuro-2a cells, antagonizing D2R effectively retarded DENV infection. Pharmacologically blocking clathrin-mediated endocytosis of DENV retarded viral replication. Targeting vacuolar-type H+-ATPase (V-ATPase)-based endosomal acidification effectively blocked the processes of DENV replication and did not have a direct effect on viral translation. The blockade of the D2R-, clathrin-, and V-ATPase-based endocytic pathways also attenuated DENV-induced neurotoxicity. Inhibiting D2R and endosomal acidification effectively retarded DENV infection, acute viral encephalitis, and mortality. These results demonstrate the D2R- and clathrin-mediated endocytosis of DENV followed by endosomal acidification-dependent viral replication in neuronal cells, which can lead to neurotoxicity.
Description:	碩士指導教授：林秋烽
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Medical Sciences] Dissertation/Thesis

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