HBV VARIANTS AND HEPATOCELLULAR CARCINOMA

乙型肝炎病毒变异体与肝细胞癌

• 批准号: 3199077
• 负责人: T. Jake Liang
• 金额: $ 16.44万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-06 至 1994-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3199077
• 关键词: enzyme linked immunosorbent assay; epitope mapping; genetic mapping; genetic polymorphism; hepatitis B antigens; hepatitis B virus group; hepatitis C virus; human subject; human tissue; laboratory mouse; liver disorder; molecular cloning; monoclonal antibody; mutant; natural gene amplification; neoplasm /cancer epidemiology; nucleic acid sequence; polymerase chain reaction; tissue /cell culture; transfection; virion; virus DNA; virus cytopathogenic effect; virus genetics; virus related neoplasm /cancer

Hepatitis B virus (HBV) infection is the most common cause of chronic liver disease on a worldwide basis and closely associated with the subsequent development of hepatocellular carcinoma (HCC). We and others have detected by monoclonal antibodies and molecular techniques HBV and/or variants in individuals with chronic liver disease and hepatocellular carcinoma previous thought to be unrelated to HBV infection. We wish to characterize these viral agents in more detail at the molecular and antigenic level, to investigate the biochemical basis of their variant biological behavior, and to evaluate their significance in the pathogenesis of HCC. We plan to do the following: 1) Evaluate the clinical and epidemiological significance of infection with low level HBV and/or variants and to assess the contribution of hepatitis C viral (HCV) infection. We will employ the polymerase chain reaction to detect, amplify and characterize low level viral sequences in serum and liver of HBsAg-negative patients with hepatocellular carcinoma. In this regard, our aims are as follows: a. Identify such agents by a rapid, sensitive and specific technique using monoclonal anti-HBs antibody to capture encapsidated virions in serum following by PCR amplification of different regions of the HBV genome. b. Use a restriction endonuclease fragment analysis following MAb capture/PCR to rapidly characterize the HBV related agents. This method will allow us to rapidly assess genomic heterogeneity. c. Demonstrate the presence of low level HBV DNA in liver of these patients and compare our results to findings in serum. d. Attempt to determine the prevalence of HCV infection by measuring the presence of antibody to HCV and HCV genome using recently available anti-HCV diagnostic kit and PCR technique, respectively. 2) Clone and sequence HBV DNA from HBsAg-negative patients in order to determine the nucleotide and amino acid variability which may be the molecular basis for their biological behavior. We also plan to explore the role of viral factor(s) in the pathogenesis of liver disease. 3) Examine the physiologic significance of genomic mutations in HBV variants in vitro in order to understand their biological properties in hepatocellular carcinoma. We will reconstruct and transfect the variant HBV genomes into human hepatoma cell lines and primary hepatocyte cultures and analyze viral antigen production and composition, transcriptional regulation, and replication competence of these variants. Based on our preliminary data obtained, we are optimistic that new information will be obtained on the clinical significance and molecular characteristics of HBV genetic variants. We believe that these agents may play a previously unrecognized role in the pathogenesis of chronic liver disease leading to or contributing to HCC. We also believe that by studying these variants in vitro and in vivo, further insights into the biological significance of genetic mutants of HBV will be obtained.

乙肝病毒感染是慢性肝病最常见的原因 世界范围内的疾病，并与随后的 肝细胞癌(HCC)的发展。我们和其他人已经发现 通过单抗和分子技术检测乙肝病毒和/或变异株 慢性肝病和肝细胞癌患者 此前被认为与乙肝病毒感染无关。我们想要描述的是 这些病毒剂在分子和抗原水平上更详细，以 调查它们不同生物学行为的生化基础，以及 探讨它们在肝细胞癌发病机制中的意义。我们计划做的是 1)评价其临床和流行病学意义 感染低水平的乙肝病毒和/或变异株，并评估 丙型肝炎病毒(HCV)感染的贡献。我们将采用 聚合酶链式反应检测、扩增和表征低水平 乙型肝炎病毒表面抗原阴性患者血清和肝脏病毒序列分析 肝细胞癌。在这方面，我们的目标如下：a. 通过一种快速、灵敏和特异的技术来鉴定此类病原体 抗-HBs单抗捕获血清中被包裹的病毒粒子 然后用聚合酶链式反应扩增乙肝病毒基因组的不同区域。B. 单抗捕获/聚合酶链式反应后进行限制性内切酶片段分析 目的：快速鉴定乙肝病毒相关病原体。这种方法将使我们能够 以快速评估基因组的异质性。C.证明存在 在这些患者的肝脏中低水平的HBVDNA，并将我们的结果与 在血清中发现。D.试图确定丙型肝炎病毒的流行率 通过检测丙型肝炎病毒抗体和丙型肝炎病毒基因组的存在而感染 最近推出的抗-HCV诊断试剂盒和聚合酶链式反应技术。 2)对HBs Ag阴性患者的HBVDNA进行克隆和测序 确定核苷酸和氨基酸的可变性可能是 它们生物学行为的分子基础。我们还计划探索 病毒因子(S)在肝病发病中的作用3)审查 乙型肝炎病毒变异株基因组突变的生理学意义 为了了解它们在肝细胞中的生物学特性 癌症。我们将重构并将变异的乙肝病毒基因组转化为 人肝癌细胞系和原代肝细胞培养及病毒分析 抗原的产生和组成，转录调控，以及 这些变体的复制能力。根据我们的初步数据 ，我们乐观地认为，将在 乙肝病毒基因的分子特征及临床意义 变种。我们认为这些特工可能扮演了之前未被认可的角色 在慢性肝病发病机制中的作用 对肝细胞癌有贡献。我们还认为，通过研究这些变种， 在体外和体内，进一步深入了解血管生成的生物学意义 将获得乙肝病毒的基因突变株。