Hepatitis C virus host interactions in micropatterened hepatocyte co-cultures

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是一个主要的世界性公共卫生问题，估计有1.2亿-1.7亿人受到影响。不幸的是，目前还不存在有效的丙型肝炎疫苗，目前的治疗方法是有毒的，而且效果参差不齐。虽然关于丙型肝炎的发病机制已经了解很多，但影响自发清除或持续感染的病毒和宿主因素仍不清楚。缺乏可靠的体外和体内模型来忠实地模拟人类自然感染的丙型肝炎病毒，继续阻碍对丙型肝炎病毒发病机制的研究。虽然肝细胞癌细胞系和永生化肝细胞使我们能够研究丙型肝炎病毒的复制，但与原代肝细胞相比，它们在表型和功能上都是异常的。虽然原代肝细胞培养被认为是研究丙型肝炎病毒感染和病毒/宿主相互作用的理想模型，但在传统培养系统中，它们的肝脏特异性功能迅速下降。最近，肝细胞和成纤维细胞的生物工程微图案化共培养(MPCCs)的产生使得在保留正常细胞功能(白蛋白产生、尿素合成等)的情况下可以长期培养肝细胞。我们研究的长期目标是更好地了解丙型肝炎的发病机制，以便设计出特异而有效的治疗方法。我们假设早期病毒和宿主的相互作用影响疾病的结果，为自发清除或慢性感染/肝纤维化奠定基础。为了深入了解肝脏对急性丙型肝炎病毒感染的早期宿主反应，我们建议表征原代人肝细胞MPCCs内和细胞复杂性增加的MPCCs内的丙型肝炎病毒/宿主相互作用。在特定目标1下，我们将在MPCCs中区分感染和未感染的细胞的表型，同时进行基因组和蛋白质组分析，以确定宿主基因和调控丙型肝炎病毒感染的途径。在具体目标2中，我们将增加MPCCs的细胞复杂性，以包括非实质细胞，以评估肝脏中的支持细胞类型是否有助于丙型肝炎病毒的发病和/或促纤维化状态的发展。在具体目标3中，我们将增加MPCCs的细胞复杂性，包括在丙型肝炎病毒感染期间滞留或招募到肝脏的淋巴细胞，以评估免疫细胞在急性丙型肝炎病毒感染中的作用。通过新型MPCCs的应用，我们可以更好地了解丙型肝炎病毒的发病机制，这可能会指导合理设计抗病毒药物的开发。有效的丙型肝炎病毒抗病毒药物或疫苗的开发将对全球公共卫生产生深远影响。

DESCRIPTION (provided by applicant): The hepatitis C virus (HCV) is a major worldwide public health problem affecting an estimated 120-170 million people. Unfortunately, an effective vaccine for HCV does not yet exist and current therapies are toxic and variably effective. While much has been learned about HCV pathogenesis, the virus and host factors that influence spontaneous clearance or persistent infection remain unknown. The lack of robust in vitro and in vivo models that faithfully mimic natural HCV infection of humans continues to hamper studies of HCV pathogenesis. Though hepatocellular carcinoma derived cell lines and immortalized hepatocytes allow us to study the replication of HCV, they are phenotypically and functionally abnormal as compared to primary hepatocytes. While primary hepatocyte cultures are thought to be an ideal model to study HCV infection and virus/host interactions, their liver specific functions rapidly decline in conventional culture systems. Recently, the creation of bioengineered micropatterned co-cultures (MPCCs) of hepatocytes and fibroblasts have allowed for long term culture of hepatocytes with the retention of normal cell function (albumin production, urea synthesis etc). The long-term goal of our research is to gain a better understanding of HCV pathogenesis in order to design specific and effective therapeutics. We hypothesize that early virus and host interactions influence the outcome of disease setting the stage for spontaneous clearance or chronic infection/liver fibrosis. To gain insight into early host response to acute HCV infection in the liver, we propose to characterize HCV virus/host interactions within primary human hepatocyte MPCCs and in MPCCs of increasing cellular complexity. Under specific aim 1, we will differentially phenotype infected and non-infected cells within MPCCs performing both genomic and proteomic profiling to identify host genes and pathways modulated in HCV infection. In specific aim 2, we will increase the cellular complexity of MPCCs to include non-parenchymal cells in order to assess if supporting cell types in the liver contribute to HCV pathogenesis and/or the development of a profibrotic state. In specific aim 3, we will increase the cellular complexity of MPCCs to include lymphocytes resident or recruited to the liver during HCV infection in order to assess the effects of immune cells on acute HCV infection. Through the use of the novel MPCCs, we may gain a better understanding of HCV pathogenesis, which may guide the development of rationally designed antiviral therapies. The development of effective HCV antivirals or vaccines would have a profound impact on global public health.