Beigel Juergen, Fella Kerstin, Kramer Peter-Juergen, Kroeger Michaela, Hewitt Philip
Molecular Toxicology, Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany.
Toxicol In Vitro. 2008 Feb;22(1):171-81. doi: 10.1016/j.tiv.2007.06.019. Epub 2007 Jul 21.
The use of animal models in pharmaceutical research is a costly and sometimes misleading method of generating toxicity data and hence predicting human safety. Therefore, in vitro test systems, such as primary rat hepatocytes, and the developing genomics and proteomics technologies, are playing an increasingly important role in toxicological research. Gene and protein expression analysis were investigated in a time series (up to 5 days) of primary rat hepatocytes cultured on collagen coated dishes. Especially after 24h, a significant down-regulation of many important Phase I and Phase II enzymes (e.g., cytochrome P450's, glutathione-S-transferases, sulfotransferases) involved in xenobiotic metabolism, and antioxidative enzymes (e.g., catalase, superoxide dismutase, glutathione peroxidase) was observed. Acute-phase-response enzymes were frequently up-regulated (e.g., LPS binding protein, alpha-2-macro-globulin, ferritin, serine proteinase inhibitor B, haptoglobin), which is likely to be a result of cellular stress caused by the cell isolation procedure (perfusion) itself. A parallel observation was the increased expression of several structural genes (e.g., beta-actin, alpha-tubulin, vimentin), possibly caused by other proliferating cell types in the culture, such as fibroblasts or alternatively by hepatocyte dedifferentiation. In conclusion, the careful interpretation of data derived from this in vitro system indicates that primary hepatocytes can be successfully used for short-term toxicity studies up to 24h. However, culturing conditions need to be further optimized to reduce the massive changes of gene and protein expression of long-term cultured hepatocytes to allow practical applications as a long-term toxicity test system.
在药物研究中使用动物模型来生成毒性数据并进而预测人类安全性是一种成本高昂且有时具有误导性的方法。因此，体外测试系统，如原代大鼠肝细胞，以及不断发展的基因组学和蛋白质组学技术，在毒理学研究中发挥着越来越重要的作用。在胶原包被培养皿上培养的原代大鼠肝细胞的时间序列（长达5天）中研究了基因和蛋白质表达分析。特别是在24小时后，观察到许多参与异源物代谢的重要I相和II相酶（如细胞色素P450、谷胱甘肽-S-转移酶、磺基转移酶）以及抗氧化酶（如过氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶）出现显著下调。急性期反应酶经常上调（如LPS结合蛋白、α-2-巨球蛋白、铁蛋白、丝氨酸蛋白酶抑制剂B、触珠蛋白），这可能是细胞分离过程（灌注）本身引起的细胞应激的结果。一个平行的观察结果是几个结构基因（如β-肌动蛋白、α-微管蛋白、波形蛋白）的表达增加，这可能是由培养物中其他增殖细胞类型（如成纤维细胞）或肝细胞去分化引起的。总之，对源自该体外系统的数据进行仔细解读表明，原代肝细胞可成功用于长达24小时的短期毒性研究。然而，培养条件需要进一步优化，以减少长期培养肝细胞基因和蛋白质表达的大量变化，以便作为长期毒性测试系统实际应用。
