Murine Model of HCV-Associated Human Liver Cancer

描述(申请人提供)：慢性丙型肝炎病毒(丙型肝炎病毒)感染现在是肝细胞癌的主要原因，肝细胞癌是一种在美国发病率迅速上升的高致命性癌症。由于缺乏丙型肝炎病毒发病的小动物模型，丙型肝炎病毒如何导致肝癌尚不清楚。这个跨学科、多PI项目的总体目标是完善最近开发的丙型肝炎人源化小鼠模型(AFC8-HU小鼠)，该模型同时拥有嵌合的人类肝脏和正常运行的人类免疫系统，并使用该模型来阐明导致丙型肝炎相关肝癌的病毒致癌机制。植入CD34+人造血干细胞和EpCAM+人肝母细胞和肝干细胞的AFC8-Hu小鼠获得~18%的肝脏嵌合率，对丙型肝炎病毒感染是允许的，并在感染丙型肝炎病毒时产生人丙型肝炎病毒特异性T细胞应答。值得注意的是，这些小鼠感染丙型肝炎病毒会导致显著的肝纤维化和氧化DNA损伤的证据--这是人类患者慢性丙型肝炎的特征。提出了三个相互关联的具体目标，以完善这一模型，并将其应用于研究丙型肝炎相关肝细胞癌的致癌机制。目的1优化方案以提高AFC8-Hu小鼠的肝脏嵌合性，并将重组DNA在细胞培养中产生的1a型HCVcc感染AFC8-Hu小鼠，从而为丙型肝炎病毒介导的致癌的反向分子病毒遗传学研究提供支持。在目标2中，在移植到AFC8小鼠体内之前，人肝母细胞中的P53将被灭活，以加速丙型肝炎病毒感染的小鼠的人肝癌的发展，并促进发现丙型肝炎病毒介导的肝癌发生的新决定因素。目的3将利用反向分子遗传学策略，在免疫介导的肝脏炎症和氧化应激的遗传毒性环境中，验证丙型肝炎病毒破坏关键抑癌功能促进癌症的假设，并具体评估丙型肝炎病毒靶向破坏视网膜母细胞瘤蛋白和丙型肝炎病毒核心蛋白封存肿瘤抑制基因DDX3所起的作用。AFC8-HU小鼠模型的进一步发展，再加上重组基因产生的感染性基因1aH77S病毒的使用，将提供一个强大而完全独特的实验系统，在其中可以阐明直接和间接致癌机制的复杂相互作用。这些研究的结果将大大增加目前对丙型肝炎病毒感染如何导致肝细胞癌发展的了解，并推动新的预防和治疗策略的开发和评估，以减轻慢性丙型肝炎患者中肝癌造成的相当大的发病率和死亡率负担。

DESCRIPTION (provided by applicant): Chronic hepatitis C virus (HCV) infection is now the leading cause of hepatocellular carcinoma (HCC), a highly lethal cancer with rapidly increasing incidence in the U.S. How HCV causes liver cancer is not well understood because of the lack of a small animal model for HCV pathogenesis. The overarching goal of this interdisciplinary, multi-PI project is to refine a recently developed humanized mouse model of hepatitis C (the AFC8-hu mouse) that possesses both a chimeric human liver and a functioning human immune system, and to use this model to elucidate viral mechanisms of carcinogenesis responsible for HCV-associated HCC. AFC8- hu mice engrafted with CD34+ human hematopoietic stem cells and EpCAM+ human hepatoblasts and hepatic stem cells achieve ~18% hepatic chimerism, are permissive for HCV infection, and develop a human HCV- specific T cell response with hepatic inflammation when infected with HCV. Remarkably, HCV infection of these mice results in significant hepatic fibrosis and evidence of oxidative DNA damage - hallmarks of chronic hepatitis C in human patients. Three interrelated specific aims are proposed to refine this model and to apply it to the study of the mechanisms of carcinogenesis responsible for HCV-associated HCC. Aim 1 focuses on optimizing protocols to enhance hepatic chimerism in AFC8-hu mice, and to infect AFC8-hu mice with genotype 1a HCVcc produced in cell culture from recombinant DNA, thereby providing a system supporting reverse molecular viral genetics studies of HCV-mediated carcinogenesis. In Aim 2, p53 will be inactivated in human hepatoblasts prior to transplantation into AFC8 mice to provide for accelerated development of human liver cancer in HCV-infected mice, and to facilitate discovery of novel determinants of HCV-mediated hepatocarcinogenesis. Aim 3 will utilize a reverse molecular genetics strategy to test the hypothesis that the disruption of critical tumor suppressor function by HCV promotes cancer in the genotoxic environment of immune-mediated hepatic inflammation and oxidative stress, and to specifically assess the role played by the targeted destruction of the retinoblastoma protein by HCV and sequestration of the tumor suppressor DDX3 by HCV core protein. Further development of the AFC8-hu mouse model, coupled with the use of infectious genotype 1a H77S virus produced from recombinant cDNA, will provide a robust and completely unique experimental system in which the complex interplay of direct and indirect mechanisms of carcinogenesis can be elucidated. The results of these studies will add significantly to current understanding of how HCV infection leads to the development of HCC, and advance the development and evaluation of new preventative and therapeutic strategies to lessen the considerable burden of morbidity and mortality imposed by liver cancer in patients with chronic hepatitis C.