Comprehensive expression analysis of primary hepatocytes under four different culture conditions

Primary hepatocytes have been widely explored as cell sources for the study of in vitro drug metabolism and pharmacokinetics (DMPK). Aiming toward establishing an in vitro drug screening method, the current study illustrated a comprehensive increase in the DMPK-related gene expression of nanopillar (NP)-cultured 3D-spheroid. To examine the expressional changes in DMPK-related genes under four different conditions, namely, NP-, sandwich (SW)-, monolayer (ML)-cultured rat hepatocytes, and freshly isolated hepatocytes, genome-wide gene-expression analysis using a DNA microarray was performed. Among the DMPK-related genes, cytochrome P450, UDP-glucuronosyltransferase, and transporter genes were focused on. Principal component analysis showed that the global gene expression profile in sample from NP culture is closer to that from freshly isolated hepatocytes than that from SW culture. The expressions of almost all Cyp 1 to 3 and Ugt genes of NP-cultured 3-D spheroid were higher than those of ML and SW. The expression of Abcc2 gene whose translation product has a critical role in excretion of metabolized bile acids in hepatocyte to bile canaliculi was three times higher in NP than in ML. From these results, 3-D spheroid formed by the NP culture was suggested to possess higher ability of metabolism and excretion than conventional 2-D monolayer culture. The NP culture has a potential as an alternative culturing technique for evaluating metabolism and toxicity toward the development of new drugs.原代肝细胞（primary hepatocytes）作为体外药物代谢与药代动力学（drug metabolism and pharmacokinetics, DMPK）研究的细胞模型已得到广泛探索。本研究旨在建立体外药物筛选方法，结果表明纳米柱（nanopillar, NP）培养的三维球体（3D-spheroid）的DMPK相关基因表达呈全面上调态势。为探究四种培养条件下大鼠原代肝细胞的DMPK相关基因表达变化，即纳米柱培养、三明治（sandwich, SW）培养、单层（monolayer, ML）培养与新鲜分离原代肝细胞组，本研究采用DNA微阵列开展全基因组基因表达分析。本次研究重点关注DMPK相关基因中的细胞色素P450（cytochrome P450）、尿苷二磷酸葡萄糖醛酸转移酶（UDP-glucuronosyltransferase）以及转运体基因。主成分分析结果显示，纳米柱培养样本的全局基因表达谱与新鲜分离肝细胞的表达谱更为接近，其相似度高于三明治培养样本。纳米柱培养的三维球体中，几乎所有Cyp 1~3家族基因与Ugt基因的表达水平均高于单层与三明治培养组。Abcc2基因的编码产物在肝细胞将代谢型胆汁酸排泄至胆小管的过程中发挥关键作用，该基因在纳米柱培养组中的表达量为单层培养组的3倍。综合上述结果，纳米柱培养制备的三维球体相较于传统二维单层培养体系，具备更优异的药物代谢与排泄能力。纳米柱培养技术有望成为新药研发中用于评估药物代谢与毒性的替代培养技术。