CW-33抑制日本腦炎病毒之功效

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题名:	CW-33抑制日本腦炎病毒之功效 The inhibition of CW-33 on Japanese Encephalitis Virus
作者:	平家鳳;Jia-Fong Ping
贡献者:	醫學檢驗生物技術學系碩士班
关键词:	CW-33;日本腦炎病毒;JAK-STAT;抗細胞凋亡;CW-33;JEV;JAK-STAT;anti-apoptosis
日期:	2013-08-02
上传时间:	2013-10-02 11:27:17 (UTC+8)
出版者:	中國醫藥大學
摘要:	日本腦炎病毒（Japanese encephalitis virus, JEV）在分類上屬於黃病毒科(Flaviviridae)，經由三斑家蚊等蚊蟲為媒介叮咬感染，感染症狀為發燒、頭痛、高燒，嚴重者急性腦膜炎、痙攣、產生神經性後遺症，甚或死亡。除預防疫苗接種外，現今對日本腦炎並無特殊有效治療方法，僅有支持性療法(干擾素治療)應用於臨床上。JEV為一單股正向RNA套膜病毒，可轉譯出一條多聚蛋白，並藉由其本身的結構蛋白及非結構蛋白，進行病毒複製組裝及干擾宿主發炎反應路徑。二氫?喃羧酸結構非常相似於?喃??，此類結構物在某些文獻中已被發現具有抗病毒及抗癌等功效，本研究中所使用之CW-33即屬二氫?喃羧酸衍生物。研究目的為鑑定CW-33抗JEV與抗細胞凋亡之功效與機轉，希望未來發展出一適合發展臨床上的安全抗病毒用藥。本實驗利用倉鼠腎細胞(Baby hamster syrian kidney cells, BHK-21)及人類腦胚胎瘤細胞(Human medulloblastoma cells, TE761)。首先為病毒複製之試管試驗，以細胞存活試驗(Cell cytotoxicity assay)觀察二氫?喃羧酸衍生物CW-33對細胞的半毒殺濃度(Concentration of 50% cytotoxicity, CC50)，對BHK-21細胞之CC50 > 500μM，而對TE671細胞之CC50為189μM。以BHK-21細胞感染JEV後，給予不同濃度的CW-33藥物處理，然後觀察細胞病變(Cytopathic effect, CPE)，取培養液測量病毒斑(Plaque)以確定病毒複製量(Virus yield)。結果發現CPE程度與病毒複製量隨著CW-33藥物濃度提升而下降。在Simultaneous-treatment、Pre-treatment、Post-treatment等三種不同給藥處理方式下，發現CW-33抑制JEV病毒斑產生的最佳作用時機為Simultaneous-treatment及Post-treatment治療模式，其達到半抑制濃度(Concentration of 50 % inhibition, IC50)為115μM。然後，經由Virus attachment及Virucidal activity assay確定CW-33抗JEV之機轉，並非為抑制感染早期時之病毒貼附細胞或影響病毒顆粒感染力。以西方墨點法(Western blotting)確認CW-33能夠磷酸化JAK-1、JAK2、Tyk2及STAT1 S727，進而活化JAK-STAT路徑。即時定量聚合?連鎖反應(Real-time PCR)發現CW-33上升調控了第一型干擾素IFNα及IFNβ和干擾素接受器IFNAR1的表現量，也提高了干擾素調節因子IRF-3、IRF-7及下游抗病毒基因PKR及OAS的mRNA生成。再者，以流式細胞儀(Flow cytometry)發現CW-33亦可抑制JEV感染所造成的細胞凋亡及調降細胞內鈣離子濃度。螢光顯微鏡觀察也發現JEV感染造成的粒線體膜電位損害因加入CW-33處理後而降低。進而確認細胞凋亡相關路徑蛋白質的表現，發現活化態的Caspase-3及Calcineurin A表現量下降，p-Akt、p-mTOR、p-ERK、p-CREB的表現量上升。本研究希望藉由鑑定CW-33藥物抑制JEV病毒感染的相關路徑機轉，其結果將可應用在治療日本腦炎之臨床用藥。 Japanese Encephalitis Virus (JEV) belongs to Flaviviridae, spreading throughout Eastern and South-eastern Asia by culicine mosquitoes, most often Culex tritaeniorhynchus. The clinical symptoms include headache, fever, vomiting, weakness, mental status changes, neurologic symptoms, movement disorders, seizures, and even death. Except for the vaccination, Japanese encephalitis has no specific treatment, just a few supportive treatments, such as IFN treatment. JEV is a single-strand positive sense RNA enveloped virus. JEV genome encodes three structural proteins (capsid, membrane, and envelope) and seven non-structural proteins, which process virus replication and disturb inflammatory responses in host cells. Dihydrofuran carboxylate CW-33 is an intermediate synthesized derivative of furoquinolines with the antiviral and anticancer activities. This study aimed to determine anti-JEV activities of CW-33 in baby hamster syrian kidney (BHK-21) cells and human medulloblastoma (TE761) cells, such cytopathic effect (CPE) inhibition, virus yield reduction, plaque reduction and apoptosis inhibition. CW-33 exhibited the less cytotoxicity with the CC50 values of > 500 μM to BHK-21 cells and 189 μM to TE671 cells, respectively. Simultaneous- and post-treatment with CW-33 alone significantly inhibited JEV-induced CPE and virus yields in BHK-21 and TE671 cells in a concentration-dependent manner. However, CW-33 had no effect on virus attachment and virucidal activity. The plaque reduction assay demonstrated CW-33 as a moderately potential anti-JEV agent with the IC50 of 115 μM. Importantly, CW-33 treatment activated JAK/STAT1 signaling pathway via elevating the phosphorylation levels of JAK1, JAK2, Tyk2, and STAT1, as well as increasing the mRNA levels of IFN-α, IFN-β, IFNAR1, IRF-3, IRF-7, PKR and OAS. The anit-JEV action of CW-33 also correlated with apoptotic reduction of JEV-infected cells, e.g. rescuing the mitochondria membrane potential, decreasing the active form of caspase-3, and increasing the phosphorylation of Akt, mTOR, ERK, and CREB. The result demonstrated CW-33 exhibiting a significant potential on the development of anti-JEV agents.
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