环境中可致肝脏损伤化学成分体外筛选模型的建立

Establishment of an in vitro Screening Model for Environmental Chemicals that Can Induce Liver Damage

环境污染日益严重,环境中的有害化学成分对人类的身体健康具有潜在的危害,而肝脏作为代谢的主要器官,无疑最易受到损伤。本研究旨在建立一种高效的、多参数的肝脏毒性体外筛选方法。采用了5种肝毒性阳性化合物和3种阴性化合物,3种试验方法对2种肝细胞进行了比较。采用四甲基偶氮唑盐微量酶反应比色法(MTT法)考察了化合物对2种细胞体外的生长抑制作用,采用高内涵检测法考察了化合物对肝损伤相关指标的影响,采用实时定量聚合酶链式反应(PCR法)比较了2种细胞的肝药酶含量。5种明确能够导致肝损伤的阳性化学物质对HepG2和L-02细胞均有不同程度的体外生长抑制作用,而3种明确不能诱导肝损伤的化合物对2种细胞均无明显生长抑制作用,且L-02细胞对肝毒性阳性化学物相对敏感。高内涵检测结果表明,不同毒性机制的阳性化合物作用于2种细胞时,与肝损伤机制相关的参数呈现出不同程度的变化,且L-02细胞预测肝损伤的精确度、灵敏度和特异度更高,表明其预测肝损伤的能力更强。此外,L-02细胞药物代谢酶的mRNA表达含量显著高于HepG2细胞,表明其与体内肝脏代谢水平更加接近。综上所述,L-02细胞结合高内涵检测为合适的肝脏毒性体外筛选模型。

The environmental pollution pose a threat to human health when exposed to harmful chemicals.Liver,mainly for metabolism,is undoubtedly the most sensitive organ to those harmful chemicals.In the present study,we established an effective in vitro screening model with multiple parameters for evaluation of hepatotoxicity.Five hepatotoxic chemicals and a plurality of three non-hepatotoxic chemicals were selected as positive controls and negative controls,respectively.Meanwhile,three kinds of assay were also applied in two hepatocytes for comparison.The growth inhibitions in these cell types induced by these chemicals were examined using MTT assay.Close relevant indicators of liver injury were then checked using high-content detection method.Hepatic enzyme contents were finally tested by real-time quantitative PCR.Five hepatotoxic chemicals showed significant inhibition on the growth of HepG2 and L-02 cells in different patterns.Furthermore,when those hepatotoxic chemicals were administrated in these cells,the liver injury-related parameters exhibited distinct degrees of changes.Our finding indicates that L-02 cells showed obvious higher sensitivity and specificity than HepG2 cells in hepatotoxic evaluation.The mRNA levels of metabolizing enzymes were also remarkably higher in L-02 cells than in HepG2 cells,indicating that L-02 cell is suitable for in vitro model closely simulating liver metabolism levels in vivo for hepatotoxic screening.