Murine Model of HCV-Associated Human Liver Cancer

描述（由申请人提供）：慢性丙型肝炎病毒（HCV）感染现在是导致肝细胞癌（HCC）的主要原因，HCC是一种高致死率的癌症，在美国发病率迅速上升。由于缺乏HCV发病机制的小动物模型，HCV如何导致肝癌尚不清楚。这个跨学科、多项目的总体目标是完善最近开发的人源化丙型肝炎小鼠模型（AFC8-hu小鼠），该模型具有嵌合的人类肝脏和功能正常的人类免疫系统，并利用该模型阐明导致丙型肝炎相关HCC的病毒致癌机制。AFC8- hu小鼠移植了CD34+人造血干细胞和EpCAM+人肝母细胞和肝干细胞，实现了约18%的肝脏嵌合，允许HCV感染，并且在感染HCV时产生人类HCV特异性T细胞反应并伴有肝脏炎症。值得注意的是，这些小鼠的HCV感染导致显著的肝纤维化和DNA氧化损伤的证据-人类慢性丙型肝炎患者的特征。提出了三个相互关联的具体目标，以完善该模型并将其应用于hcv相关HCC的致癌机制研究。目的1：优化方案，增强AFC8-hu小鼠的肝脏嵌合，并用重组DNA细胞培养产生的基因型1a型HCVcc感染AFC8-hu小鼠，从而为hcv介导癌变的反向分子病毒遗传学研究提供系统支持。在Aim 2中，在移植到AFC8小鼠之前，p53将在人肝母细胞中失活，以加速hcv感染小鼠的人肝癌发展，并促进发现hcv介导的肝癌发生的新决定因素。Aim 3将利用反向分子遗传学策略来验证HCV破坏关键抑癌功能在免疫介导的肝脏炎症和氧化应激的遗传毒性环境中促进癌症的假设，并具体评估HCV靶向破坏视网膜母细胞瘤蛋白和HCV核心蛋白隔离肿瘤抑制因子DDX3所起的作用。AFC8-hu小鼠模型的进一步发展，加上重组cDNA产生的传染性基因型1a H77S病毒的使用，将提供一个强大的和完全独特的实验系统，可以阐明直接和间接致癌机制的复杂相互作用。这些研究的结果将显著增加当前对HCV感染如何导致HCC发展的理解，并推进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因肝癌造成的严重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.