- 學位論文
利用新建立之C型肝炎病毒感染細胞株篩選可調節病毒複製之宿主細胞因子及化合物
Selection of host factors and chemicals that regulate the replication of hepatitis C virus by using a newly established infectious clone
摘要
C型肝炎病毒（hepatitis C virus, HCV）屬於黃熱病毒科（Flaviviridae）正向單股RNA病毒。基因體約9.6 kb，能夠產生結構蛋白質core、E1、E2 、p7以及非結構蛋白質NS2、NS3、NS4A、NS4B、NS5A、NS5B。由於C型肝炎病毒RNA複製時的突變率很高，使C型肝炎病毒能逃過宿主的攻擊。C型肝炎病毒主要是經由血液或體液感染，感染後約有80％的病患換轉變成慢性肝炎，其中約有10-20％病患會產生肝硬化，也有約1-5％的病患會轉變為肝癌。全球約有超過1.7億的人口感染C型肝炎病毒，已成為了一個必須積極去面對與解決的全球性健康問題。 本實驗室希望能夠建立一個利用Huh7細胞產生C型肝炎病毒的穩定表現系統。本研究首先將質體pJFH1進行改造，在C型肝炎病毒基因後端再接上D型肝炎病毒的ribozyme和poly A signal，完成質體pJFH1-RZ-PA的建構。將質體pJFH1-RZ-PA轉染到Huh7細胞中，兩天後萃取細胞內的RNA進行RT-PCR，結果能夠偵測到反股的C型肝炎病毒RNA，也能偵測到核心蛋白質的表現，顯示藉由轉染送入的質體所產生的C型肝炎病毒正股RNA能夠自行複製，產生反股RNA。再將質體的T7驅動子置換成CMV 驅動子，完成質體pCMV-JFH1-RZ-PA之建構，再用質體pCMV-JFH1-RZ-PA轉染Huh7細胞，在G418存在下，挑選出能夠穩定表現HCV RNA的Huh7細胞株Huh7-CMV9。 由先前的論文指出，hnRNP（heterogeneous nuclear ribonucleoprotein） family的成員會和C型肝炎病毒RNA結合，幫助C型肝炎病毒的複製。我們利用shRNA技術以及穩定表現HCV RNA的Huh7細胞株Huh7-CMV9，研究hnRNP哪一些成員參與C型肝炎病毒的複製。結果發現 hnRNP P2/ FUS若被knockdown，會使細胞中的HCV反股RNA量減少，顯示hnRNP P2可能參與C型肝炎病毒的複製。 為了尋找抗C型肝炎病毒藥物，利用Huh7-CMV9複製系統以測試藥物對進行C型肝炎病毒於細胞中所累積的反股RNA量的影響。結果顯示從48種藥物之中有10種藥物可抑制C型肝炎病毒於細胞中所累積的反股RNA量一半以上。進一步實驗結果測得藥物D46的IC50濃度為3.43μM。hnRNP P2/ FUS和藥物D46如何調節C型肝炎病毒RNA複製之詳細作用機轉，須再進一步研究。
並列摘要
Hepatitis C virus (HCV) infects about 170 million people worldwide and has been a major public health problem. HCV contains a positive-stranded RNA genome of 9.6 kilobases that encodes a polyprotein of about 3000 amino acid residues. The majority of HCV-infected patients fail to clear the virus and develop chronic liver diseases including cirrhosis and hepatocellular carcinoma. For screening chemicals and cellular factors that can inhibit HCV replication, an infectious HCV clone was first established, plasmid pCMV-JFH1-RZ-PA that contains sequences representing the full-length genomic RNA of HCV under the control of CMV promoter, followed by the ribozyme of hepatitis delta virus was constructed. Huh7 cells were transfected with pCMV-JFH1-RZ-PA and pCMV-Tag2 containing neomycin-resistant gene. Following G418 selection, one clone, designated Huh7-CMV9, that is capable of producing viral RNA and proteins at high levels was identified. In addition, HCV-like particles were detected in the Huh7-CMV9 cells by Electron Microscopy. Heterogeneous nuclear ribonucleoprotein A1 was recently shown to be important for HCV replication. By introducing shRNAs into the Huh7-CMV9 cells in this study, hnRNP P2 was identified to specifically reduce the replication of HCV. On the other hand, 10 out of 48 chemicals examined showed inhibitory effects on HCV replication in the Huh7-CMV9 system. Among these chemicals, the IC50 of chemical D46 to inhibit HCV replication is 3.43μM. How hnRNP P2 and D46 regulate HCV replication requires further studies.