以 C6/36 與 293T 細胞探討四環黴素衍生物對登革熱病毒二型及日本腦炎病毒繁衍之影響

Abstract

登革熱是目前影響全球健康的重要議題之一，主要分布在熱帶及亞熱帶地區。根據統計已超過 100 個國家曾經爆發過登革熱疫情並且約有二十五億人口受到登革熱病毒的威脅。登革熱病毒屬於黃質病毒科黃質病毒屬。對於登革熱目前並沒有有效且商業化之疫苗，積極開發抗登革熱藥物是當前重要目標之一。 黃質病毒的外膜蛋白對於病毒與宿主細胞結合，進入宿主細胞內都扮演著很重要的角色。之前實驗室已根據登革熱病毒外膜蛋白篩選出一些四環黴素衍生物在幼倉鼠細胞 ( BHK-21 ) 有抑制登革熱病毒二型空斑產生的效果。在本研究中，我使用蚊子細胞 ( C6/36 ) 及 人類胚胎腎臟細胞 ( 293T ) 進行一系列實驗，藉由實驗結果推測四環黴素衍生物對登革熱病毒抑制效果的標的。結果顯示使用不同細胞株不會影響四環黴素衍生物對登革熱病毒的抑制效果。初步推斷四環黴素衍生物之抑制標的應在病毒上。 為了進一步了解四環黴素衍生物抑制的專一性，使用同為黃質病毒的日本腦炎病毒 ( Japanese encephalitis virus ) 與藥物一起感染 293T 細胞。結果得到除 Chlortetracycline 之外其他四環黴素衍生物對日本腦炎病毒並沒有顯著抑制的效果，顯示四環藥物衍生物中 Tetracycline、Doxycycline、Rolitetracycline 的抑制效果具有專一性。

Dengue virus, a member of the Flaviviridae family, is now believed to be the most important arthropod-borne disease in the world, especially in tropical and subtropical regions. There were outbreaks reported in more than 100 countries and about 2 billion people lived under the threats. Therefore, control of the dengue viral infections is an urgent issue. However, there is no commercial vaccine or medication available. Dengue envelop protein (E protein) is responsible for activating “membrane fusion” during the entry of viruses into host cells. Previously research in the laboratory has shown that several tetracycline derivatives have inhibitory effect on the propagation of dengue viruses in mammalian cell BHK21. In this study, I used mosquito cell line C6/36 and human embryonic cell line kidney 293T to perform a series of assay to find out the target of these tetracycline derivatives. All these tetracycline derivatives showed inhibitory effects on dengue virus type 2 in both C6/36 and 293T cell culture, indicating that the inhibitory target might be at virus instead of the host cell. Furthermore, to understand the specificity of these tetracycline derivatives, Japanese encephalitis virus (JEV), which is also a member of flavivirus was used to infect 293T cell line with or without the present of these four tetracycline derivatives. All these tetracycline derivatives showed no significant inhibitory effects on JEV in 293T cell line except chlortetracycline.