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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24h及48h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 alternative model of non-clinical safety evaluation of carcinogenicity of primary mouse hepatocytes and to investigate the potential mechanism of action of genotoxic and non-genotoxic carcinogens by toxicological genomics. Methods: The primary mouse hepatocyte sandwich culture model was constructed by modified two - step collagenase perfusion method to separate C57BL / 6 mouse hepatocytes. Primary mouse hepatocytes were given two genotoxic carcinogens, aflatoxin B1 (AFB1), benzo (a) pyrene, BAP and two non-genotoxic carcinogen thioacetamide After co-culturing with thioacetamide (TAA) and pirinid (WY-14643, WY) for 24h and 48h, Affymetrix mouse transcriptome analysis gene chip 1.0 (GeneChip ~? Mouse Transcriptome Assay 1.0) analysis. The differentially expressed genes were further studied on the possible mechanism of carcinogens using gene enrichment (GEP) enrichment analysis, KEGG pathway functional analysis, principal component analysis (PCA) and hierarchical clustering analysis (HCA). Results: The number of differentially expressed genes in genotoxic carcinogens was higher than non-genotoxic carcinogens. Although AFB1 and BAP affect chemical carcinogenic pathway and up-regulate p53 signaling pathway, their carcinogenic pathways are different. TAA affects oxidoreductase activity and may cause oxidative damage. WY significantly up-regulates peroxisome proliferator-activated receptors and lipid synthesis and metabolic pathways, possibly resulting in disruption of intracellular lipid metabolism. More information can be drawn for further analysis by extending the carcinogen exposure time. Conclusion: The in vitro model of primary mouse hepatocyte sandwiches was successfully established and could be used to study the mechanism of genotoxic carcinogens and non-genotoxic carcinogens. It is expected to be one of the candidate surrogate candidates for non-clinical safety evaluation of carcinogenicity in vitro.