臺灣博碩士論文加值系統

B型肝炎病毒蛋白HBx一直被懷疑與肝癌的形成有很大關係，但詳細的機制尚未釐清。本論文主要是想研究HBx蛋白對p53蛋白的功能，細胞存活及HPRT基因突變率之影響。本研究是利用已建立的可受tetracycline調控之HBx可誘導性表達系統的HepG2-3X肝癌細胞株作為實驗模型。在實驗中發現HBx蛋白會促使細胞中的p53蛋白停滯於細胞質中，而且受紫外線活化的p53(Ser-15)蛋白之磷酸化現象會受到抑制。之後，利用p53報告基因活性實驗分析p53的轉活化能力，發現當HBx蛋白存在時，細胞中的p53之轉活化能力減低了50 % 。在總體性基因修補分析實驗中，當HBx蛋白存在時，不論細胞遭受單次或重複性紫外線照射，其移除損傷產物的能力皆受到阻礙，而且在有HBx蛋白時會提高細胞對紫外線傷害之敏感性。接著以MTS方法分析HBx蛋白是否會影響細胞對抗癌藥物cisplatin、adriamycin及taxol的敏感性。在HBx蛋白存在下，細胞對cisplatin及adriamycin的敏感性降低，而對taxol的敏感性則提高。在cisplatin處理下細胞p53蛋白磷酸化顯著增加，反之，p53蛋白磷酸化不受taxol影響。HBx蛋白似乎會減少細胞HPRT基因突變率，此部份結果及其作用機轉還有待進一步研究。

The hepatitis B virus X protein (HBx) is thought to be involved in the development of hepatocellular carcinoma (HCC) , but its exact role in HCC remains unclear. The purpose of this study was to investigate the effect of HBx on p53 function and the role of the HBx in cell survival and mutation frequency in HPRT gene in response to DNA damage. HepG2-3X hepatoma cell line that express HBx in a tetracycline inducible system was established previously in our laboratory and was applied for this study. When HBx protein was expressed in HepG2-3X, cytoplasmic retention of p53 protein was enhanced where level of phospho-p53 (Ser-15) protein activated by UV irradiation was abrogated. The p53-mediated transactivation ability was decreased to about 50 % in the presence of HBx protein in a reporter assay. Global genomic repair (GGR) assay showed that removal of DNA photo-products in the cells under either one or repeated UV exposure was reduced in the presence of HBx. Treatment of cells with UV or taxol resulted in decreased cell survival when HBx was expressed. In contrast, cells that expressed HBx were more resistant to cisplatin or adriamycin. p53 phosphorylation at serine-15 was increased in response to cisplatin treatment, but it remained unchanged when taxol was used. In the cells that expressed HBx, mutation frequency in HPRT gene was reduced. This result was unexpected and the reason for this is not clear at the present time.

目 錄
英文名詞對照表························3
中文摘要···························4
英文摘要···························5
緒論·····························6
一、B型肝炎病毒·······················6
二、HBx蛋白質························7
三、腫瘤抑制因子p53·····················8
四、核甘酸切除修護系統NER··················11
五、Tet-off gene expression system·············12
六、HPRT基因與基因突變頻率·················13
七、研究目的························14
實驗方法···························16
一、細胞的培養與處理····················16
二、反轉錄-聚合鏈反應·················17
三、西方墨點分析法·····················18
四、細胞質與細胞核蛋白之分布················20
五、p53蛋白之磷酸化····················21
六、p53轉活化能力分析···················22
七、總體性基因修補分析···················24
八、細胞存活率分析·····················26
九、HPRT基因突變分析····················27
十、統計方法························28
實驗結果···························29
討論·····························37
參考文獻···························45
圖表說明···························55
附錄·····························78
誌謝·····························85

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