建立原代小鼠肝细胞体外模型用于致癌物作用机制研究

目的：建立药物非临床安全性评价致癌性试验原代小鼠肝细胞体外替代模型，以毒理基因组学方法对遗传毒性致癌物和非遗传毒性致癌物的潜在作用机制进行研究。方法：改良两步胶原酶灌流法分离C57BL/6小鼠肝细胞构建原代小鼠肝细胞三明治培养模型。原代小鼠肝细胞分别给予2个遗传毒性致癌物黄曲霉素B1（aflatoxin B1，AFB1）、苯并芘[benzo（a）pyrene，BAP]和2个非遗传毒性致癌物硫代乙酰胺（thioacetamide，TAA）、匹立尼酸（WY-14643，WY）共培养24 h及48 h后，使用Affymetrix公司小鼠转录组分析基因芯片1.0（GeneChip^® Mouse Transcriptome Assay 1.0）进行时间依赖差异表达基因分析。对差异表达基因使用基因功能（GO）富集分析，KEGG通路功能分析，主成分分析（PCA）和分层聚类分析（HCA）等进一步研究了致癌物可能的作用机制。结果：遗传毒性致癌物的差异表达基因的数量比非遗传毒性致癌物多。AFB1和BAP虽然影响化学性致癌通路和上调p53信号通路，但其致癌途径有一定差异。TAA影响氧化还原酶活性，可能造成氧化损伤。WY显著上调过氧化物酶体增殖物激活受体和脂肪合成与代谢通路，可能造成细胞内脂质代谢紊乱。通过延长致癌物暴露时间能得出更多信息用于进一步分析。结论：成功建立了原代小鼠肝细胞三明治体外模型，可用于研究遗传毒性致癌物和非遗传毒性致癌物的作用机制，有望作为药物非临床安全性评价致癌性试验体外替代候选模型之一。

Objective: To establish an in vitro primary mouse hepatocyte model as an alternative method for carcinogenicity study of drugs in the field of nonclinical safety evaluation of drugs, and investigate potential modes of action of genotoxic and non-genotoxic carcinogens using toxicogenomics methods. Methods: Primary mouse hepatocytes were isolated from C57BL/6 male mice using the modified two-step collagenase perfusion method and cultured in a collagen-collagen sandwich configuration. The cells were exposed for 24 and 48 hour with two genotoxic carcinogens aflatoxin B1(AFB1)and benzo(a)pyrene(BAP)and two non-genotoxic carcinogens thioacetamide(TAA)and WY-14643(WY)to study time-dependent gene expression profiling using the GeneChip^® Mouse Transcriptome Assay 1.0 from Affymetrix Company. The differentially expressed genes were used for gene ontology(GO)enrichment analysis, KEGG pathway analysis, principle component analysis(PCA)and hierarchical clustering analysis(HCA)to study their possible carcinogenic mechanisms. Results: The genotoxic carcinogens had more differentially expressed genes than non-genotoxic carcinogens. Although both AFB1 and BAP regulated chemical carcinogenesis and up-regulated p53 signaling pathway, the carcinogenic pathway had some differences. TAA influenced the activity of oxidoreductase, which may cause oxidative damage. WY significantly up-regulated PPAR signaling pathway and fat synthetic and metabolic pathways, leading to the result of lipid metabolism disturbance. More information was available for further analysis with elongation of exposure time. Conclusion: An in vitro sandwich-cultured primary mouse hepatocyte model was successfully established, which could be used to study the potential modes of action of genotoxic and non-genotoxic carcinogens and might be a promising candidate in vitro model for carcinogenicity study in the field of nonclinical safety evaluation of drugs.