2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643317摘要：去氧核醣核酸甲基化與組織胺修飾在調控B型肝炎病毒共價環狀去氧核醣核酸活性上所扮演的角色: 對慢性B型肝炎病程可能造成的影響慢性B 型肝炎感染會造成不同程度的病程，輕微如不活動性肝炎，但有的會造成慢性活動性肝炎，導致肝硬化或肝癌。慢性肝炎的病程已知和病毒複製活性有關。B 型肝炎病毒的複製，主要是由B 型肝炎病毒共價環狀去氧核醣核酸(簡稱共價環狀閉合DNA)所控制，共價環狀閉合DNA 是B 型肝炎病毒複製的模板。目前抗病毒藥物無法根除B 型肝炎，主要的原因就是因為此種DNA 即使在抗病毒藥治療之下，還可以存在病人受感染的肝細胞內相當久。表顯遺傳修飾，包括組織蛋白(Histone)修飾以及DNA 甲基化，會調控真核細胞的基因轉錄。最近研究發現表顯遺傳會影響B 型肝炎病毒基因表達與複製。所以此研究目的在探討慢性B 型肝炎病患，其B 型肝炎病毒共價環狀閉合DNA 是否發生DNA 甲基化或組織蛋白修飾，以及此種變化是否影響病程。我們也將利用新發展的B 型肝炎病毒體外感染模型，去研究DNA 甲基化和組織蛋白修飾如何影響共價環狀閉合DNA。此研究有四個主要目標: (1)研究共價環狀閉合DNA 甲基化程度，並了解是否與B 型肝炎e 抗原陰轉有關。我們發展出從受感染肝臟組織中純化共價環狀閉合DNA並分析其甲基化程度的方法，我們將利用慢性B 型肝炎病人的肝臟組織進行此一研究。(2)深入且全面性分析不同病程的慢性B 型肝炎病人，其共價環狀閉合DNA 甲基化程度(全基因體)(3)研究組織蛋白修飾對共價環狀閉合DNA 複製能力的影響(4)利用B 型肝炎體外感染模型(HepaRG cells)，研究表顯遺傳如何調控共價環狀閉合DNA 的基因表達。我們相信了解共價環狀閉合DNA 的表顯遺傳調控，將能洞察B 型肝炎病毒的複製機轉，並幫助發展新治療方式，去靜默化B型肝炎複製，以長期抑制或根除慢性B 型肝炎病患體內長存的共價環狀閉合DNA。<br> Abstract: The role of DNA methylation and histone modification in the replicativeactivity of HBV covalently closed circular DNA: implications in the diseasestatus of chronic hepatitis BChronic hepatitis B virus (HBV) infection causes a wide spectrum of disease status, ranging from aninactive carrier state to chronic active hepatitis, liver cirrhosis and even hepatocellular carcinoma. It hasbeen demonstrated that the clinical outcomes of chronic hepatitis B is strongly associated with thereplicative activity of HBV. HBV replication is primarily controlled by the activity of a particular form ofreplicative template, the covalently closed circular DNA (cccDNA), which is clinically important, becauseit forms a major barrier to eradication of persistent HBV from infected individuals by current antiviraltherapy. Epigenetic alternations, including histone modifications and DNA methylation, are known toregulate the transcriptional activity of eukaryotic genomes. Recently, it has been shown that epigeneticmodulation may inhibit HBV replication by silencing the HBV gene transcription. Therefore, the goal ofthis project is to determine the DNA methylation and histone modification in the HBV cccDNA and theircorrelation with the disease status of chronic hepatitis B patients. An in vitro HBV infection system willalso be utilized to explore the underlying mechanisms of epigenetic regulation. Four specific aims will bepursued: (1) To correlate the methylation patterns of HBV cccDNA with the status of HBeAgseroconversion. HBeAg seroconversion of chronic hepatitis B patients is often associated with thereduction of HBV replication and improving hepatic injury. In our lab, we have developed a protocol thatcan specifically and quantitatively analyze the methylation patterns of HBV cccDNA. The methylationpatterns and levels of the HBV cccDNA isolated from the liver tissues of chronic hepatitis B patients will beanalyzed and correlated with the status of HBeAg seroconversion.(2) To perform a comprehensive andquantitative analysis of methylation patterns of HBV cccDNA in HBV-infected patients with differentdisease status, including inactive carrier state, tolerant stage, chronic active hepatitis , and cirrhosis.To further quantitatively determine the relationship between the HBV cccDNA methylation and the diseasestatus, we will utilize the next-generation sequencing strategy to quantitatively analyze the methylationpatterns of the full-genome HBV cccDNA. (3) To investigate the effects of histone modification on thetranscriptional activity of each individual promoter of HBV cccDNA. The minichromosome of HBVcccDNA will be isolated using the sucrose gradient centrifugation. The histone modifications of the HBVcccDNA minichromosome will be determined using the chromatin Immunoprecipitation (ChIP) approachand correlated with HBeAg-seroconversion and disease status. (4) To investigate the mechanisms of DNAmethylation and histone modification in regulation of HBV cccDNA replicative activity using an invitro HBV infection model (HepaRG cells) An in vitro HBV infection system using HepaRG cells will beestablished. Using this model, we will analyze the effects of epigenetic modulations on the replicativeactivity of HBV cccDNA. We believe that understanding the epigenetic regulation of HBV cccDNA can notonly provide deep insight into the mechanisms of viral replication, but also may help design novel therapyto silence viral replication and eventually lead to the long-term suppression or even eradication of thepersistent cccDNA in chronic hepatitis B patients.慢性B型肝炎B型肝炎病毒共價密環去氧核醣核酸e抗原陰轉DNA甲基化Chronic hepatitis BHepatitis B viruscccDNAHBeAg seroconversionDNA methylation