臺灣博碩士論文加值系統

慢性B型肝炎病毒感染是人類最嚴重的病毒性感染之一。B 型肝炎病毒持續存在於患者體內，其主要原因在於病毒持續性的以共價密環去氧核醣核酸（cccDNA）的形式存於慢性B型肝炎病毒患者的肝細胞核中，而一般相信cccDNA為B型肝炎病毒基因體的轉錄模板。Nucleoside 類似物─(-)-2'', 3''-dideoxy-thiocytidine（lamivudine; [3TC]），是一種通過驗證的口服用藥，可有效的抑制患者 HBV DNA 的複製。然而，目前已知 HBV DNA 會在停藥病人血清中快速的重現。由體外組織培養細胞的研究，推測 cccDNA 是 HBV DNA 再現的主因。此外，目前沒有任何一種藥物可真正治癒慢性 B型肝炎病毒患者。為了進一步了解 HBV cccDNA 持續性（persistence），將含有 BclI遺傳標誌的 1.3 倍 HBV 基因體質體轉染 HepG2 細胞後，以RT-PCR分析（假如 HBV mRNAs 是從 cccDNA 轉錄而來，BclI遺傳標誌將出現在其所有 mRNA 3’端），我們得到了兩種細胞株：第一類細胞株（細胞株 1.3.ES11），其所有 HBV mRNA 3’ 端皆具有BclI遺傳標誌；第二類細胞（細胞株 1.3.ES2、1.3.ES8），其 HBV mRNA 3’ 端有些具有 BclI遺傳標誌，有些則否。以南方墨點法分析染色體DNA，這些細胞株都含有完整的 1.3 倍 HBV 基因體。細胞株 1.3.ES2、1.3.ES11含有一個 integreted site，而細胞株 1.3.ES8 則有兩個。以 BclI 限制酶及南方墨點法分析細胞株 1.3.ES11 染色體 DNA，得到大約 3.2 kb HBV 片段。此結果支持其 1.3倍 HBV 基因體 3’ 端含有 BclI 遺傳標誌。南方墨點法分析細胞株1.3.ES2、3.3.ES8的 Hirt supernatant 時，根據 EcoRI 切割及有 heat danaturation抵抗性，可以指出其兩種細胞株都可產生 cccDNA。
我們已經建立了可產生 HBV cccDNA 的細胞株，而第二類細胞株將被使用於進行實驗，藉此觀察在 proliferating cell 或 arrest cell 中，lamivudine 處理與否對其 ccc DNA 持續性是否有影響。在未來，希望能以這些體外細胞培養的方法，對 cccDNA 進行更細部的研究。

Chronic hepatitis B (CHB) is one of the most serious viral infections in humans worldwide. Virus persistence in CHB patient relies on maintaining a pool of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes, which presumably is the template of HBV gene expression. (-)-2’, 3’-thiacytidine (lamivudine; 3TC), an approved oral drug, is a nucleoside analogue which effectively inhibits the replication of HBV in CHB patients. However, it has been showed that cessation of drug treatment resulted in rapid rebound of HBV DNA in the patients’ sera. In vitro studies suggested that the cccDNA might be responsible for the reappearance of HBV DNA. To get insight into the understanding of the persistence of the HBV cccDNA, two classes of HBV producing lines, based on the RT-PCR analysis were established by transfecting BclI-bearing 1.3-fold HBV genome into HepG2 cells (The BclI site would present on the 3’end of all HBV mRNAs if their transcribed from cccDNA but not from introduced DNA template). Class one cell (clone 1.3.ES11) is characterized by containing BclI site at the 3’end at all HBV mRNAs. In the class two cell (clone 1.3.ES2 and 1.3.ES8), the produced mRNAs are mixture products in which the 3’end of HBV mRNAs either contained BclI site or did not. Southern blot analysis of the genomic DNA suggested that all clones contained intact 1.3-fold HBV genome with one integrated site (clone 1.3.ES2 and 1.3.ES11) or two sites (1.3.ES8). However, BclI restriction and Southern analysis of 1.3.ES11 genomic DNA generated an HBV-containing fragment with size approximately 3.2 kb, suggesting that the 3’end of the 1.3-fold HBV genome contain a BclI site. This finding may account for the fact that the 3’ end of all HBV mRNAs derived from 1.3.ES11 cells contains the BclI site. Southern analysis of Hirt supernatants prepared from 1.3.ES2 and 1.3.ES8 cells clearly indicated that this two cell lines produced cccDNA, as evidenced by band shifting after digesting with EcoRI and resistant to heat denaturation.
We have established HBV cccDNA-producing lines. Class two cells are being used to assess the effects of proliferating or arrested cells treated with or without lamivudine on the maintenance of cccDNA. In the next, the more detailed study of cccDNA might be accomplished in this cell culture system.

英文摘要-------------------------------------------------------1
中文摘要-------------------------------------------------------2
壹、緒論-------------------------------------------------------3
貳、材料與方法-------------------------------------------------8
參、結果------------------------------------------------------17
肆、討論------------------------------------------------------24
伍、參考文獻--------------------------------------------------29
附表及附圖----------------------------------------------------35

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