B型肝炎病毒為部分雙股的DNA病毒，慢性B型肝炎患者罹患肝癌的機率是正常人的100倍以上。B型肝炎病毒含有三種形式的表面抗原，全長的表面抗原基因包含pre-S1、pre-S2及S區域 (region)，大型表面抗原是由表面抗原基因的第一個起始碼 (start codon) 所轉譯出來的，包含pre-S1、pre-S2及S；中型表面抗原是由表面抗原基因的第二個起始碼 (start codon) 開始轉譯出來的，包含pre-S2及S；小型表面抗原是由表面抗原基因的第三個起始碼 (start codon) 開始轉譯出來的，僅含有S部分。pre-S的刪除型突變 (deletion mutant)導致這些突變型抗原累積在肝細胞的內質網中，形成毛玻璃狀肝細胞(ground glass hepatocytes；GGH)，並引發強烈的內質網壓力。我們的實驗目標就是去探討pre-S突變型的表面抗原是否引發基因體的不穩定性。我們利用H2DCF (2’7’-dichlorofluorescein diacetate) 染色及彗星分析 (comet assay) 證實了當細胞存有pre-S突變型的表面抗原時，引發較高量的反應性氧化物質 (reactive oxygen species；ROS) 和氧化性的DNA損傷，即是8-oxoguanine的量在基因體中明顯的增加許多。而這些氧化性的DNA損傷也活化了DNA修復系統中的鹼基切除修復途徑 (base excision repair)。另外在隨機基因突變試驗 (random gene mutation assay) 中發現pre-S突變型的表面抗原引發的基因突變率較wild-type的表面抗原基因高。另外pre-S2 mutant的轉殖基因老鼠中發現疑似有p53突變 (p53 muatant) 發生，而DNA repair基因8-oxoguanine glycosylase (OGG1) 也受刺激而上升 (up regulation)。我們更進一步利用臨床上因受HBV感染而引發肝癌 (HBV related HCC) 的病人肝臟組織，利用雷射捕獲顯微分離技術 (LCM) 將表現GGH type II的肝細胞收集下來，並進行RT-PCR觀察OGG1之表現量，結果發現OGG1的表現量也受到刺激而上升 (up regulation)。這表示著這些HCC病人的肝臟細胞中持續存在氧化性的DNA損傷 (oxidative DNA damage)，而這些氧化性的DNA損傷引發基因體的不穩定，因此可能與癌症的發生有關。

Hepatitis B virus (HBV) is a small DNA virus with partially double-stranded genome. Chronic HBV infection have a greater than 100-fold increased relative risk of developing the tumor. There are three forms of the HBV surface protein. The large form is translated from the first ATG of the ORF; while the middle and small forms are translated from downstream in-frame ATG codons. The pre-S deletion mutants of HBS antigen accumulated in hepatocytes reveal ground-glass hepatocytes (GGH), which are often seen in patients with chronic HBV infection. Accumulation of this mutant protein in endoplasmic reticulum (ER) induces an oxidative stress. We aimed to study the potential genomic instabilities caused by the pre-S mutant HBS antigen. By the DCFH-DA stainings as well as the comet assays, the Huh-7 cells over-expressing the pre-S mutant HBS antigens exhibited higher levels of reactive oxygen species (ROS) and oxidative DNA lesion 8-oxoguanine. These oxidative DNA damages have significantly stimulated the base excision repair (BER) activity in cells. In addition, the assays of random gene mutation frequencies using hprt gene as a marker have found that the pre-S mutant HBS antigens induce higher mutation rates as compared with the wild-type large HBS antigen does. In the transgenic mice of the pre-S2 HBS antigen, we observed that the tumor suppressor gene p53 was mutated and the DNA repair gene 8-oxoguanine glycosylase (OGG1) was up-regulated. The OGG1 gene was also induced in the hepatic tissues harvested by laser capture microdissection in the HCC patients, indicating that the oxidative DNA damages occur in the HCC cells with chronic HBV infection. These oxidative DNA damages probably induce genomic instability, which potentially lead to hepatocellular carcinogenesis.

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