中文摘要
慢性B型肝炎病毒(hepatitis B virus, HBV)感染的病人是肝癌發生的高危險群，但HBV如何引起肝癌，至今仍尚未澄清。我們實驗室先前在毛玻璃肝細胞(ground glass hepatocyte, GGH)中發現有兩種大表面蛋白突變株，其基因在pre-S序列有片段缺失(deletion)，分別稱為pre-S突變株I型(∆S1)及II型(∆S2)。Pre-S突變株已知位於肝細胞內質網，可以引起內質網壓力、基因的不穩定，以及調控cyclin A及cyclooxygenase-2基因，而引起細胞不正常的生長。在肝組織中，含∆S2的GGHs常呈群聚生長，其原因有待澄清。過去的研究指出，生長因子(growth factor)在肝癌的病程中扮演很重要的角色。因此，pre-S突變株是否可以調控生長因子及與肝癌發生的關係須進一步釐清。在本研究中，我們首先篩選幾個已知的肝細胞相關生長因子，再以Affymetrix基因微矩陣的技術來尋找HuH-7細胞中受∆S1及∆S2調控的生長因子基因及其受體。再以即時聚合酵素連鎖反應(real-time PCR)及酵素免疫偵測法(ELISA)來確認。結果發現，表皮生長因子(EGF)、纖維母細胞生長因子(FGF)、轉型生長因子(transforming growth factor beta-1, TGF-beta 1)及以上三者的受體(receptors)都可受到pre-S突變蛋白的調控。因TGF-beta在肝癌的發生扮演重要角色，所以本研究乃進一步研究pre-S突變株對TGF-beta及其受體的調控。結果發現，野生型或pre-S突變蛋白都會增強TGF-beta 1的表現及分泌量。但只有表現∆S2突變蛋白的肝癌細胞株，可以抗拒TGF-beta 1引起的細胞週期停止。且∆S2突變蛋白也可選擇性地降低TGF-beta 1受體(TGF beta R-II)的表現。因此，雖然HBV大表面蛋白都會增強TGF-beta 1的表現，但只有∆S2突變蛋白可以選擇性降低TGF beta R-II的表現，此一發現可能與含∆S2的GGHs常呈群聚生長或生長優勢相關。此一機制進一步支持pre-S突變蛋白在肝癌發生上所扮演的角色。更深入的分子機制及信息傳導路徑值得進一步去釐清。

Abstract
Individuals infected by hepatitis B virus (HBV) are at a high risk for the development of hepatocellular carcinoma (HCC). However, how HBV promotes the development of HCC remains unclear. Previously, two types of HBV large surface protein (LHBs) mutants, pre-S1 (△S1) and pre-S2 (△S2) were identified in ground glass hepatocytes (GGHs) in our laboratory and have been reported to induce ER stress, genomic instability, and regulate cyclin A and cyclooxygenase2. All these effects can induce abnormal cell growth which may contribute to the development of HCC. In the literatures, cytokines and growth factors have been reported to play a significant role in the progression of HCC. Whether these growth factors may play a role in the pre-S-mutant-induced hepatocarcinogenisis remain to be clarified. In this study, we first used Affymetrix array analysis to investigate the regulation of growth factors by pre-S mutant proteins in HuH-7 cells. The mRNA and protein expression of growth factors was further confirmed by real-time PCR and ELISA test. The data revealed the expression of epidermal growth factor, fibroblast growth factor, transforming growth factor beta-1 (TGF-β1), and their receptors are selectively regulated by pre-S mutants. Since TGF-β1 may lead to hepatocyte apoptosis, we next examined whether TGF-β1 would affect HuH-7 growth in the presence of pre-S mutant proteins. We found that HuH-7 cells stably expressed △S2 LHBs were more resistant to exogenous TGF-β1 induced cell-cycle arrest. Furthermore, △S2 LHBs could selectively down-regulate the expression of TGF-β receptor II (TGFβR-II). The regulation of TGF-β1 and TGFβR-II by △S2 mutant may relate to clonal expansion or growth advantage of GGHs containing △S2 mutant. This result further supports the role of pre-S mutants in HBV-related hepatocarcinogenesis. The detailed molecular mechanism of HBV pre-S mutants in the progression of HCC is worth to be investigated in the future.

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 陳建仁，楊懷壹，未發表資料
 張仲明，未發表資料