摘要
C型肝炎病毒(HCV)是造成輸血後肝炎的主要病原，其感染分佈現已成全球化趨勢，根據世界衛生組織統計，全世界已有超過3%以上人口受HCV感染，也就是說全球有一億七千萬人感染HCV。HCV感染後，約有80%急性C型肝炎病患會轉變為慢性C型肝炎，其中20%會繼續惡化成肝硬化，最後約有1-4%病人會發展成肝癌。目前臨床上較有效的療法是採用甲型干擾素與Ribavirin的合併治療，但也只有約40%病患有療效，也就是說更有效的治療策略仍有待研發。至於疫苗的發展亦是困難重重，導因於病毒基因的突變率太高，甚至無法保護再次受到同一病毒感染。HCV屬Flaviviridae科、Hepacivirus屬，病毒顆粒外包被有套膜，基因體約為9.6 kb的正股RNA，僅具一段開放性讀架，可先轉譯出一段約含3010個胺基酸的多蛋白前驅物，之後再經宿主細胞的訊號蛋白和病毒本身的蛋白切割出10個成熟的病毒蛋白：C、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B。NS2為23 kD的越膜蛋白，被認為是NS5A磷酸化所必需，但直到現在，科學家對它在肝細胞中的功能仍所知甚微，本研究的最終目標即想了解，NS2在HCV的致病過程中扮演什麼角色，故藉由cDNA microarray雜交方法分析轉染pEGFP-N1和pNS2-2-EGFP的肝癌細胞株HuH-7中，由C型肝炎病毒NS2蛋白所誘發的基因表現差異。將瞬時轉染的HuH-7以G418 (500 µg/mL)篩選培養，挑選出穩定轉染細胞株，含有pEGFP-N1的細胞株便定名為N1-H01，而擁有pNS2-2-EGFP的就稱作NS2-H01，之後便以PCR偵測NS2基因，RT-PCR和Western blotting檢測NS2-EFGP的RNA和蛋白質表現，在PCR和RT-PCR的實驗結果，均可在NS2-H01中發現600 bp特異性線帶出現，而在Western blotting中亦可見50 kD特異性線帶顯現，NS2-EGFP的持續表現亦可由NS2-H01中微弱的綠色螢光得到印證。在cDNA microarray分析結果中，CD9位居mRNA上升表達基因之首，而在FACS的實驗數據中，亦可見CD9表現量在NS2-H01有被增加調控，但CD81卻被降低調控。CD9和CD81皆屬tetraspanin superfamily一員，其特徵為具有四段越膜區段，有研究報告指出，CD9可與pro-HB-EGF結合，因而改變其構型，造成細胞生長速率加快，於是便分析MTT assay，發現NS2-H01細胞株的確長得比N1-H01快，這與先前研究報告結果相吻合。總之，HCV NS2可在HuH-7中增加調控CD9和降低調控CD81表現量，除此之外，HCV NS2亦可增快肝癌細胞株的生長。本實驗顯示出HCV NS2蛋白具有調節肝細胞的功能，而此發現將有助於未來HCC的相關研究。

Abstract
Hepatitis C virus (HCV) is the major etiological agent of post-transfusion hepatitis and widespread globally. According the World Health Organization survey, up to 3% of the world's population being infected with HCV, it suggests that more than 170 million people worldwide be infected by HCV. About 80% of patients with acute HCV infection progress to chronic hepatitis, 20% of these to cirrhosis, and 1 to 4% to hepatocellular carcinoma (HCC). The current therapy available is combinational treatment with alpha interferon and ribavirin. That only 40% of all patients beneficial from this treatment to develop a sustained response calls the urgent need for more effective antiviral therapeutics. Vaccine development has been hampered by a high degree of antigenic variation and the lack of protection against viral reinfection. HCV has been classified as the sole member of a distinct genus called Hepacivirus in the Flaviviridae family. The enveloped virion has a positive-stranded linear RNA genome of approximately 9.6 kb and a large open reading frame encoding a precursor polyprotein of about 3010 amino acids. This polyprotein is cleaved by host cellular signalase(s) and viral proteases into 10 mature viral proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The NS2, a 23kD transmembrane polypeptide, has shown to be required for NS5A phosphorylation. However, little is known about the effects of NS2 on human liver cells. The specific aim of this study is to investigate the role of NS2 protein in the pathogenesis of HCV. cDNA microarray hybridization was exploited to characterize the differentially expressed genes induced by HCV NS2 in the hepatoma cell lines HuH-7 with the transfection of pEGFP-N1 and pNS2-2-EGFP. The transiently transfected HuH-7 cells were selected by G418 (500 µg/mL) to obtain stably cell lines carrying pEGFP-N1 and pNS2-2-EGFP, which were designed as N1-H01 and NS2-H01 cells. The existence of NS2 gene was demonstrated by PCR and the expression of NS2-EFGP was analyzed with RT-PCR as well as western blotting by RNA and protein. The PCR and RT-PCR resulted in 600 bp specific bands in NS2-H01 cells and the western blotting demonstrated a 50 kD specific band. The constitutively NS2-EGFP expression was also evidenced by the emitted green fluorescence light in NS2-H01 cells. In cDNA microarray assay, CD9 was on top of the list by mRNA level elevated in NS2-H01 cells. FACS analysis showed the up-regulation of CD9 molecules but the down-regulation of CD81 molecules in NS2-H01 as compared to N1-H01 and HuH-7 cells. Both CD9 and CD81 belong to tetraspanin superfamily by the characterization of the four conserved transmembrane segments. CD9 has been shown to bind to pro-HB-EGF (precursor of heparin-binding-epidermal growth factor) and modify the conformation of pro-HB-EGF, which might boost the cellular growth rate. According to the MTT assay, NS2-H01 had a higher growth rate than N1-H01, which agrees with the evidence of the previous report. In conclusion, HCV NS2 up-regulated CD9 and down-regulated CD81 expression in HuH-7 cells. In addition, HCV NS2 facilitated the growth of hepatoma cells. This study reveals the effects of HCV NS2 protein on hepatocyte modulation, with the relevance to HCC remaining further investigation.

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