出版年:

2000

研究生:

林銀玲

研究生(英文姓名):

YIN-LING LIN

論文名稱:

登革二型病毒感染人類肝細胞及被Carboxyfullerene抑制的探討

英文論文名稱:

Study on Dengue-2 Virus Infection and Prevention by Carboxyfullerene in Human Liver Cells

指導教授:

劉校生

指導教授(英文姓名):

Hsiao-Sheng Liu

學位類別:

博士

校院名稱:

國立成功大學　

系所名稱:

基礎醫學研究所

學號:

S58831079

學年度:

89

語文別:

英文

論文頁數:

88

關鍵詞:

肝細胞 ; 登革二型病毒 ; 活化態氧複合物

英文關鍵詞:

liver cells ; Dengue-2 virus ; Carboxyfullerene ;
Sodium butyrate ; RANTES ; NF-IL-6 ; ROS ;
envelope

被引用次數:

0

[ 摘要 ]

登革病毒感染會引發致死性的出血性及休克性症候群 (DHF/DSS)，其病人被發現有肝功能異常的情形。為了了解登革病毒和肝細胞的關係，我們建立登革病毒感染人類肝細胞的系統來加以探討。我們不僅分析病毒直接感染肝細胞所產生的傷害，也同時去偵測感染之肝細胞所產生的免疫因子，並進而用藥物來抑制登革病毒的感染。首先我們證實登革病毒可以感染五株不同特性的肝細胞株並引發細胞的病變。進一步比較之間的差異發現登革病毒在分化較好的細胞(如Huh7, PLC, Hep3B and Chang liver)有較好的複製能力，並造成程度不等的細胞傷害。反之在分化較差的細胞(如HA22T)其病毒複製則相對較弱。利用Sodium butyrate 來促進HA22T的細胞分化，可以增加登革病毒的感染，顯示分化相關的因子可能在登革病毒感染肝細胞中扮演著重要的角色。另一方面，我們發現登革病毒感染肝細胞會引發一個驅動因子RANTES (regulated upon activation, normal T cell expressed and secreted) 的產生。進一步研究它的調控機轉，我們發現登革病毒感染肝細胞會活化RANTES的基因轉錄，其中介於RANTES起動子+1到-181之間有二個蛋白質結合位置(NF-IL-6和NF-kB)分別會被活化的NF-IL-6以及一個未知的蛋白質所結合。利用抗氧化物N-acetyl-L-cystein (NAC), Nitro-L-argininie methyl ester (L-NAME) and pyrrolidine dithiocarbamate (PDTC)可以抑制登革病毒感染所活化的RANTES之基因表現以及NF-IL-6結合DNA的能力，因而推論oxidants參與NF-IL-6的活化並進而引發RANTES基因的表現。此外，進一步利用carboxyfullerene (C60)可以抑制登革病毒感染肝細胞並阻止病毒所引起的細胞傷害。研究其抑制病毒的機轉，我們發現C60的C3異構體可以在沒有光激發的情況下有效抑制病毒入侵細胞。從比較不同極性的C60異構體，以及分析病毒有無Envelope對C60的感受性，我們證實C60的C3異構體可以透過與脂質的極性作用抑制具有Envelope的病毒。總而言之，本論文發現登革病毒感染分化較好的肝癌細胞具有較高效率的病毒複製以及較嚴重的細胞病變，並證實登革病毒感染可以活化RANTES基因之表現並研究其調控的機轉，將有助於了解肝細胞在DHF/DSS中所參與的角色。此外我們發現C60具有須光和不須光的二種機制來抑制病毒，可以拓展C60 將來用藥的廣泛性。

[ 英文摘要 ]

Liver malfunction has been diagnosed in the patients with the syndrome of dengue hemorrhagic fever and/or shock syndrome (DHF/DSS). To clarify the relationship between dengue virus and the liver malfunction, we used liver cell culture to investigate whether dengue-2 virus infection could cause cell damage and induce the production of immune regulators, as well as be inhibited by antiviral agents. Firstly, we show that dengue-2 virus replication accompanied with various severities of cytopathic effects (CPE) in the differentiated liver cells such as HuH-7, PLC, Hep3B and Chang, but poorly in the dedifferentiated cells such as HA22T. Differentiation related factors seem to play an important role in dengue virus replication and this speculation was clarified by a differentiation inducer sodium butyrate. Moreover, we demonstrated that the secretion of chemokine RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) was activated in the infected liver cells. Our data demonstrated that RANTES expression was induced at transcription level in dengue-2 virus infected liver cells, and NF-IL-6 and an undefined factor other than NF-kB were found to form DNA-protein complex on the RANTES promoter region (+1 to -181) in dengue virus infected cells. We used antioxidants N-acetyl-L-cysteine (NAC), Nitro-L-arginine methyl ester (L-NAME) and pyrrolidine dithiocarbamate (PDTC) to further clarify that NF-IL-6 and RANTES might be in the same pathway and the oxidants were probably the up-stream regulator of RANTES expression. To prevent dengue virus infection, we used carboxyfullerene (C60) in our study. C60 is known as a photosensitizer for virus inactivation. C60 with C3 symmetry, named the C3 isomer, could also inactivate dengue-2 virus without light. Further analysis discovers a light independent pathway, in which the C3 isomer suppression of enveloped viruses is affected through its hydrophobic interaction with the viral lipid envelope. In summary, our study demonstrated the direct effect and the induced RANTES expression by dengue-2 virus infection in human liver cells would pave the way for better understanding the liver pathogenesis involved in DHF/DSS. Moreover, we find that the light-dependent and -independent mechanisms of C60 for inactivation of dengue virus will broaden the application of C60 in the future.