臺灣博碩士論文加值系統

B型肝炎病毒 (Hepatitis B virus, HBV) 是人類重要的感染性疾病之一，慢性的B型肝炎病毒感染通常會導致慢性肝炎 (chronic hepatitis) 甚至併發肝硬化(liver cirrhosis)，並且與肝癌(Hepatocellular carcinoma)的發生關係密切。在急性感染及慢性感染的B型肝炎病患中常可發現B型肝炎病毒突變種，許多突變種主要是在慢性感染的特定階段中出現。B型肝炎病毒的突變種中，C基因的缺損突變可在一些特定病患群中被測得，本研究將探討B型肝炎病毒C基因的缺損突變的發生率，並尋找B型肝炎病毒C基因的缺損突變發生後對疾病造成的可能影響。我們初步收集慢性B型肝炎、肝硬化、肝癌等病患的血清並純化其 DNA，利用 PCR 的方式進行B型肝炎病毒C基因 DNA的擴增反應， 以探討B型肝炎病毒C基因的缺損發生率，並對此基因序列進行更進一步的研究。目前已發現在肝癌病人，而非肝炎或肝硬化病人中有一可造成聚合酶過度表現的C基因缺損突變種，透過多種實驗分析發現此突變種會加速細胞增生；且表現此突變種相較於沒有表現此突變的肝癌細胞，較不易發生細胞凋亡；另外在裸鼠實驗發現此突變種較易引發皮下腫瘤的形成。進一步的實驗發現此突變種蛋白會與 miR-10b 以及 miR-183 發生交互作用。

Hepatitis B virus (HBV) is one of the most important human pathogens in chronic infectious diseases. Chronic hepatitis B virus infection can lead to chronic active hepatitis and liver cirrhosis, which further results in emergence of hepatocellular carcinoma. Naturally occurring mutations in HBV have been found in patients with acute or chronic HBV infection, wherein some mutants develop in certain stages of chronic HBV infection. Of these HBV mutations, core-gene-defective mutants have been found in some special patient groups. Our present study aims to investigate the prevalence of core-gene-defective HBV mutants in different patient groups as well as the possible pathological effect of the mutants in chronic HBV infection. Firstly, we collected serum samples from patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. To understand the prevalence of core-gene-defective HBV mutants in these three groups of patients, HBV DNA was extracted for PCR amplification of the core genes followed by nucleotide sequencing. We have found a core-gene-defective mutant which causes polymerase overexpression. This type of mutants was presence in patients with hepatocellular carcinoma, but not in patients with chronic hepatitis or liver cirrhosis. Cell growth assay revealed enhanced cell proliferation in hepatoma cells expressing the mutant protein. A lower percentage of apoptotic cells was found in hepatoma cells carrying the mutant HBV, compared to the wild type. Tumorigenecity experiments showed that hepatoma cells carrying the mutant HBV more easily developed xenograft tumors in nude mice. Further studied revealed that the mutant protein physically interacted with miR-10b and miR-183, the miRNAs targeting at many growth enhancing genes.

目錄
指導教授推薦書
口試委員會審定書
授權書 iii
誌謝 vi
中文摘要 viii
英文摘要 ix
目錄 x
第一章 前言 - 1 -
1.1 B型肝炎病毒 (Hepatitis B virus, HBV)簡介 - 1 -
1.2 C基因的詳細介紹及重要性 - 3 -
1.3 C基因的缺損 - 4 -
1.4 先前研究及研究動機 - 8 -
第二章 材料與方法 - 9 -
2.1 萃取病人血清的 DNA - 9 -
2.2 聚合酵素鏈鎖反應 (The Polymerase Chain Reaction, PCR) - 9 -
2.3 DNA膠體電泳分析 - 10 -
2.4 DNA膠體純化 - 10 -
2.5 轉型 (transformation) - 11 -
2.6 純化菌液中的 DNA - 11 -
2.7 質體構築 (plasmid construction) - 12 -
2.8 細胞培養(cell culture) - 14 -
2.9 轉染(transfection) - 14 -
2.10 免疫螢光染色 (Immunofluorescence assay) - 15 -
2.11 建立穩定細胞株 - 15 -
2.12 (MTT) assay - 16 -
2.13 TUNEL assay - 17 -
2.14 利用 Nude mice進行in vivo實驗 - 17 -
2.15 Soft agar assay - 18 -
2.16 純化蛋白質 - 18 -
2.17 免疫沉澱法 (Immunoprecipitation assay) - 18 -
2.18 萃取RNA - 19 -
2.19 miRNA反轉錄 - 19 -
2.20 Quantitative PCR - 20 -
第三章 結果 - 21 -
3.1 B型肝炎病毒C基因的缺損突變在三組B型肝炎病人中的發生率 - 21 -
3.2 質體構築 (plasmid construction) - 21 -
3.3 免疫螢光染色標定C–P片段缺損基因 - 22 -
3.4 測試穩定細胞株建立成功 - 23 -
3.5 缺損C–P基因對於細胞生長的影響(in vitro) - 23 -
3.6 缺損C–P基因對細胞凋亡的影響 - 24 -
3.7 缺損之C–P片段基因於裸鼠體內的影響 - 25 -
3.8 DNA聚合酵素與miRNA的交互作用 - 25 -
第四章 討論 - 27 -
參考資料 - 31 -
圖 - 37 -
表 - 47 -
附錄 - 48 -

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