Development of 3D Differentiated Hepatocyte Culture Systems for the Study of HCC

描述（由申请人提供）：丙型肝炎病毒（HCV）引起急性和慢性肝炎和肝细胞癌（HCC）。值得注意的是，HCV在HCC的上升中发挥了重要作用，在美国占50%的病例。然而，尽管有明显的需求，但没有疫苗可用于预防丙型肝炎病毒感染，而且只有一小部分慢性感染患者对目前的治疗方案有反应。阻碍HCV研究的主要障碍是缺乏细胞培养和小动物感染模型。尽管我们和其他研究人员发现基因型2a共识克隆可以在人肝癌来源的Huh7细胞中体外产生感染性HCV，已经取得了重大进展，但仍然需要一个生理学上相关的分化肝细胞培养模型来阐明HCV诱导的肝细胞功能改变如何导致HCV相关的肝脏疾病，特别是病毒在肝细胞恶性转化中的作用。因此，本提案的目的是建立一个人类肝组织模型，用于研究HCV感染和HCV相关HCC发展的分子细节。具体来说，nasa设计的生物反应器，被称为旋转壁血管（RWVs），将用于建立一个低剪切，低湍流的环境，允许细胞在空间上共定位，三维生长，并分化成高度特化的组织。需要验证的假设是，在旋转壁血管中培养的肝细胞在功能上与体内的肝脏更相似，因此这些细胞将为研究HCV发病机制，特别是HCC提供一个改进的系统。因此，本研究的具体目的是：1)在RWV中对3d培养的Huh7细胞进行表征，以确定是否可以诱导这些肝癌细胞分化并下调其癌症特异性标志物。2)在RWV中三维建立原代人肝细胞培养物，并表征其是否保持分化表型。3)评估和使用三维Huh7和原代人肝细胞培养来研究HCV-宿主细胞相互作用和由此产生的细胞病理学，特别是HCV相关HCC的病因学。公共卫生相关性：全球丙型肝炎病毒（HCV）感染人数超过1.7亿，占美国肝细胞癌（HCC）病例的50%。由于需要一个生理上相关的非转化、分化的肝细胞组织培养模型来阐明病毒在肝细胞恶性转化中的作用，本提案的总体目标是建立一个先进的人类肝组织模型，用于研究HCV感染和HCV相关HCC发展的分子细节。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) causes acute and chronic hepatitis and hepatocellular carcinoma (HCC). Notably, HCV has played a major role in the rise of HCC, accounting for 50% of cases in the United States. Despite an obvious need however, no vaccine is available to protect against HCV infection and only a subset of chronically infected patients respond to current treatment options. The major obstacle impeding HCV research has been a lack of cell culture and small animal infection models. While significant advancement has been made with the identification of a genotype 2a consensus clone that we and others have shown can produce infectious HCV in vitro in human hepatoma-derived Huh7 cells, a physiologically relevant, differentiated hepatocyte culture model is still needed to elucidate how HCV-induced alterations in hepatocyte function lead to HCV-associated liver disease, specifically the role the virus plays in malignant transformation of hepatocytes. As such, the objective of this proposal is to develop a human liver tissue model for the study of the molecular details involved in HCV infection and the development of HCV-associated HCC. Specifically, NASA-engineered bioreactors, termed rotating wall vessels (RWVs), will be utilized to establish a low-shear, low-turbulence environment that allows cells to co-localize spatially, grow three-dimensionally, and differentiate into highly specialized tissues. The hypotheses to be tested are that hepatocytes cultured in rotating wall vessels are functionally more similar to the liver in vivo and that these cells therefore will provide an improved system to study HCV pathogenesis, particularly HCC. Accordingly, the specific aims of this proposal are: 1) Characterize 3-D cultured Huh7 cells in the RWV to determine if these hepatoma cells can be induced to differentiate and down regulate their cancer-specific markers. 2) Establish primary human hepatocyte cultures three-dimensionally in the RWV and characterize whether they maintain a differentiated phenotype. 3) Evaluate and use three-dimensional Huh7 and primary human hepatocyte cultures for the study of HCV- host cell interactions and resulting cellular pathology, particularly the etiology of HCV-associated HCC. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) infects more than 170 million people worldwide, accounting for up to 50% of cases of hepatocellular carcinoma (HCC) in the United States. Because a physiologically relevant non-transformed, differentiated hepatocyte tissue culture model is needed to elucidate the role the virus plays in malignant transformation of hepatocytes, the overall objective of this proposal is to develop an advanced human liver tissue model for the study of the molecular details involved in HCV infection and the development of HCV-associated HCC.