Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes
Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes
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DOI：
10.1124/dmd.108.025775
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发表时间：
2009-06-01
影响因子：
3.9
通讯作者：
van Delft, Joost H. M.
中科院分区：
文献类型：
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作者：
Mathijs, Karen;Kienhuis, Anne S.;van Delft, Joost H. M.
原代人和大鼠肝细胞培养物是毒理学研究中使用的体外系统。然而，虽然转基因小鼠模型提供了一个机会，研究机制的毒性，小鼠原代肝细胞培养的描述较少。在这项研究中，通过研究异生物质代谢和基因表达谱的时间依赖性能力，评估了基于小鼠肝细胞的体外模型的潜在有用性。原代小鼠肝细胞，使用两步胶原酶灌注分离，培养在胶原蛋白三明治配置。基因表达谱和各种细胞色素P450（P450）酶的活性测定后0，42，90小时的文化。基因表达谱的主成分分析显示每个时间点的重复是相似的。大多数I期生物转化酶的基因表达水平在42和90小时分别下降到原始水平的约69和57%，而大多数研究的P450的酶活性下降到59和34%。II期基因表达的下降仅为96和92%的原始水平，分别在42和90小时。途径分析揭示了蛋白质、外部信号通路和能量产生水平的初始效应。后期的影响，观察转录，翻译，膜，细胞周期相关的基因集。这些结果表明，体外培养的原代小鼠肝细胞系统是稳健的，似乎比大鼠肝细胞系统更好地保持其代谢能力。
Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using two-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various cytochrome P450 (P450) enzymes were determined after 0, 42, and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are similar. Gene expression levels of most phase I biotransformation enzymes decrease to approximately 69 and 57% of the original levels at 42 and 90 h, respectively, whereas enzyme activities for most of the studied P450s decrease to 59 and 34%. The decrease for phase II gene expression is only to 96 and 92% of the original levels at 42 and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways, and energy production. Later effects are observed for transcription, translation, membranes, and cell cycle-related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and seems to maintain its metabolic competence better than that of the rat hepatocyte system.