臺灣博碩士論文加值系統

全世界約有超過一億七千萬的人口感染丙型肝炎病毒 ( Hepatitis C virus ; HCV )，而導致病人產生急性和慢性肝疾病。丙型肝炎病毒是一種具有套模的病毒，帶有約9.6kb單股的RNA。它屬於黃熱病毒科 ( Flaviviridae )。目前帶有丙型肝炎病毒的病人唯一有效治療方式為干擾素α和 ribavirin 的合併治療。尚未有有效的疫苗，而標準的治療方式( 長效型干擾素α加上ribavirin ) 也只有對50–60% 的病人是有效的，而且通常也會伴隨著嚴重的副作用。環胞黴素A ( Cyclosporin A ; CsA ) 是一個廣泛被使用的免疫抑制劑，已有文獻發表可以有效對抗HCV的感染。環胞黴素A的抗病毒作用是藉由阻斷細胞內環胞黴素A的結合蛋白親環素 ( cyclophilins )。因此，細胞因子會影響丙型肝炎病毒的複製，因此可以做為抗病毒的標的。在這個研究中，我們要去尋找可以幫助丙型肝炎病毒複製的細胞因子。利用微陣列分析尋找在 HCV replicon 細胞株和 mutant 的細胞株 ( 沒有 HCV replicon ) 基因表現的差異性。部分表現有差異性的基因，再利用 RT-qPCR 做更進一步的證實。再利用 shRNA 技術個別去抑制在 HCV replicon 細胞株表現較多的基因，然後去測定是否影響丙型肝炎病毒的複製；丙型肝炎病毒的複製是利用監測丙型肝炎病毒 NS5A 蛋白的表現量來決定。我們發現細胞因子 KCNJ8 可以幫助丙型肝炎病毒的複製。之後我們使用它的抑制劑 PNU-37883A 去阻斷它的表現並且偵測丙型肝炎病毒 NS5A 蛋白的表現量，我們發現 KCNJ8 會影響丙型肝炎病毒的複製。此外， KCNJ8抑制劑合併干擾素或環胞黴素A ，比干擾素或環胞黴素A單獨處理更有抑制丙型肝炎病毒複製的效果。這個結果顯示 KCNJ8 有潛力當作限制丙型肝炎病毒複製的抗病毒標的。

Hepatitis C virus ( HCV ) infects more than 170 million people worldwide, leading to both acute and chronic liver diseases in patients. Hepatitis C virus ( HCV ) is an enveloped virus with a single stranded 9.6-kb RNA genome. It is a member of the Flaviviridae. Nowadays, the combined therapy with interferon-alpha ( IFN-α ) and ribavirin ( RBV ) remains the only available option for treatment of patients with chronic hepatitis C. There is no vaccine available and the standard therapy [ ( pegylated ) interferon alfa plus ribavirin ] is only effective in 50–60% of patients and is associated with important side-effects. Cyclosporin A ( CsA ) , a widely used immunosuppressive drug, has been reported to be effective against HCV infection. The antiviral action of CsA is mediated by blockade of actions of cellular CsA-binding proteins, the cyclophilins. Therefore, cellular cofactors affecting hepatitis C virus replication could be the potential anti-viral targets. In this study, we are going to search for the cellular factors that can facilitate HCV replication. Microarray analysis was used to screen the genes with differential expression level between the HCV replicon cell line and mutant cell line without HCV replicon. Some of these differentially expressed genes were further verified by RT-qPCR. Genes over-expressed in HCV replicon were knocked down by shRNA technology individually to determine their effect on the HCV replication. The replication of HCV was monitored by the expression of HCV NS5A protein. The cellular factor KCNJ8 was identified. Then, we used its inhibitor PNU-37883A to block its expression and detected the expression of HCV NS5A protein. We found that KCNJ8 could affect HCV replication. Moreover, the combination treatment of either interferon-α and PNU-37883A or CsA and PNU-37883A could inhibit HCV replication more effectively than that of interferon-α or CsA treatment alone. These results indicate that KCNJ8 could be a potential antiviral target to limit HCV replication.

緒論……………………………………………………………………………………1
丙型肝炎病毒……………………………………………………………………1
丙型肝炎之流行病學與臨床症狀…………………………………………2
丙型肝炎臨床診斷…………………………………………………………3
丙型肝炎的治療……………………………………………………………4
干擾素…………………………………………………………………5
Ribavirin……………………………………………………………….6
環孢素(Cyclosporin A ,CsA)………………………………………….7
KC NJ8 ( potassium inwardly-rectifying channel, subfamily J, member 8 )………9
實驗材料與方法……………………………………………………………………..11
細胞培養………………………………………………………………………..11
RNA萃取……………………………………………………………………….12
西方點墨法....................................................13
ECL呈色…………………………………………………………………13
AP呈色…………………………………………………………………..14
Lentiviral Vector 系統…………………………………………………………14
Lentiviral production……………………………………………………..15
Lentiviral infection……………………………………………………….16
反轉錄聚合�○s鎖反應………………………………………………………..17
即時定量聚合�○s鎖反應……………………………………………………..17
細胞存活率測試………………………………………………………………..18
藥品試劑………………………………………………………………………..19
西方點墨法使用的抗體………………………………………………………..21
實驗流程……………………………………………………………………………..22
實驗結果……………………………………………………………………………..23
Part 1：分析HCV replicon 與突變株mut’3 細胞株間基因表現的差異性…23
Part 2 ：尋找幫助HCV複製的細胞因子……………………………………24
Part 3：KCNJ8的抑制劑可抑制丙型肝炎病毒複製…………………………26
Part4：KCNJ8的抑制劑分別與Cyclosporin A 或Interferon-α 作用的效果.....28
討論.................................................................... 29
參考文獻..........................................................50

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