A Comparative Study of Genome Wide Transcriptional Profiles of Primary Hepatocyte in In Vitro Cultures

The liver is one of most important organs in our bodies. It performs many essential functions including metabolism, synthesis, secretion, detoxification, and storage. Hepatocytes are the principal cell type in the liver and are involved in multiple liver-specific functions. There have been several efforts to develop in vitro culture systems capable of maintaining hepatocyte-specific phenotype over long time periods. In hepatic tissue engineering, two commonly used culture systems are the collagen sandwich and monolayers of cells. In this study, genome-wide gene expression profiles of primary hepatocytes were measured over an 8-day period for each cell culture system using Affymetrix GeneChips and analyzed via Gene Set Enrichment Analysis (GSEA), which is a powerful method to elicit biologically meaningful information from microarray data at the level of gene sets. Results indicate that the gene expression in hepatocytes in collagen sandwich cultures gradually diverges from that in monolayer cultures. Gene sets up-regulated in collagen sandwich cultures include those associated with liver metabolic and synthetic functions. These functions are associated with lipid, amino acid, carbohydrate, and alcohol metabolism and bile acid synthesis. Nuclear receptors are up-regulated in collagen sandwiches 24 hours after seeding. Signals transmitted from these receptors may cause the up-regulation of other processes in subsequent days. Cytochrome-P450 monooxygenase expression was initially down-regulated but exhibited up-regulation after 72 hours. Our results provide a baseline for further explorations into the systems biology of engineered liver mimics as well as 2D and 3D co-cultures of primary hepatocytes and non-parenchymal cells.肝脏是人体最为重要的器官之一，其具备代谢、合成、分泌、解毒与储能等多项核心生理功能。肝细胞（hepatocytes）是肝脏的主要细胞类型，参与多种肝脏特异性生理活动。此前已有多项研究致力于开发可长期维持肝细胞特异性表型的体外培养体系。在肝脏组织工程领域，两类常用的细胞培养方案为胶原三明治培养体系（collagen sandwich）与单层细胞培养体系。本研究中，研究者针对两种培养体系中的原代肝细胞（primary hepatocytes），在8天的培养周期内通过Affymetrix基因芯片（Affymetrix GeneChips）获取全基因组基因表达谱，并借助基因集富集分析（Gene Set Enrichment Analysis, GSEA）进行数据分析——该方法是从基因集层面挖掘微阵列数据中生物学关联信息的高效手段。结果表明，胶原三明治培养体系内的肝细胞基因表达模式会逐渐与单层培养体系产生显著分化。在胶原三明治培养体系中上调的基因集，多与肝脏代谢及合成功能相关，具体涵盖脂质、氨基酸、碳水化合物与乙醇代谢以及胆汁酸合成通路。核受体在细胞接种后24小时即在胶原三明治培养体系中出现上调，其介导的信号传导或在后续培养天数中引发其他生物学过程的上调表达。细胞色素P450单加氧酶（Cytochrome-P450 monooxygenase）的表达最初呈现下调趋势，但在培养72小时后转为上调。本研究结果可为后续探索工程化肝脏模拟物，以及原代肝细胞与非实质细胞（non-parenchymal cells）的二维、三维共培养体系的系统生物学研究提供基准参照。