三维HepaRG细胞模型的建立及对曲格列酮肝毒性评价  

Establishment of three-dimensional HepaRG cell model and assessment of troglitazone-induced hepatotoxicity

作　　者：张弛 李凤祥[2] 彭辉 王韫芳 赵君 郭家彬 彭双清 ZHANG Chi;LI Feng-xiang;PENG Hui;WANG Yun-fang;ZHAO Jun;GUO Jia-bin;PENG Shuang-qing(Academy of Military Medicine y Academy of Military Sciences y Beijing 100850,China;Center of Disease Control and Prevention,PLA,Beijing,China;Military Hospital of the 13rd Army Group,Xiamen 361000,China)

机构地区：[1]军事科学院军事医学研究院,北京100850 [2]解放军疾病预防控制中心,北京100071 [3]中国人民解放军73集团军医院,厦门361000

出　　处：《毒理学杂志》2020年第2期109-114,共6页Journal of Toxicology

基　　金：国家重研发计划课题(2018YFC1602602);国家自然科学基金(81430090);北京市科技新星项目(Z171100001117103)。

摘　　要：目的利用人肝癌HepaRG细胞系建立3D肝细胞模型,并应用该模型对抗糖尿病药物曲格列酮所诱导的肝毒性进行评价。方法采用低吸附法构建3D HepaRG细胞/组织模型。3D HepaRG细胞给予曲格列酮(3.125、6.25、12.5、25和50μmol/L)处理不同时间后,应用Alamar blue法测定细胞存活率,高内涵成像分析检测线粒体活性氧自由基、线粒体膜电位和线粒体丰度,以评价曲格列酮引起的肝细胞损伤。结果以1000或3 000个细胞/孔密度接种细胞,细胞在第3天自发聚集形成致密的球形,第7天后可形成稳定球体。曲格列酮可剂量依赖性地降低3D HepaRG细胞的存活率,诱导3D细胞线粒体损伤,表现为线粒体活性氧自由基生成增加,线粒体膜电位和线粒体丰度下降。结论采用低吸附法成功构建了3D HepaRG模型并应用该模型阐明曲格列酮诱导的肝毒性特征,提示3D HepaRG模型在评估药物性肝损伤等肝毒性测试中具有潜在的重要应用价值。Objective To establish three-dimensional(3 D) spherical models of HepaRG cells and evaluate the hepatotoxicity of troglitazone, a widely used drug to treat non-insulin dependent-diabetes. Methods The 3 D culture of HepaRG cells was established by ultra-low attachment method. Afterward, the cultures were exposed to troglitazone at the concentrations of 3.125, 6.25, 12.5, 25, and 50 μmol/L. Cell viability was determined using alamar blue method. To evaluate troglitazone-induced mitochondrial toxicity, mitochondrial reactive oxygen species(ROS), mitochondrial membrane potential, and mitochondrial mass were evaluated by high-content imaging analysis(HCA). Result At the seeding density of 1000 or 3000 cells, the cells appeared to spontaneously aggregate to form compact spherical units on the day 3 in 3 D culture after seeding and produced spheroids with diameters of approximately 200 to 300 μm on the 7 th day. The cell viability of 3 D cultures was significantly decreased by troglitazone in a concentration-dependent and time-dependent manner. Troglitazone induced mitochondrial damage in 3 D cultures was evidenced by enhanced ROS accumulation, mitochondrial membrane potential loss, and reduction of mitochondrial mass. Conclusion The 3 D HepaRG model was successfully established by ultra-low attachment method. By using this model, it illustrated that the characteristics of hepatic toxicity of troglitazone and suggested that 3 D HepaRG cultures are promising models in the assessment of chemical induced liver toxicity such as drug-induced liver injury.

关 键 词：3D模型 曲格列酮 药物性肝损伤 线粒体损伤 高内涵分析 

分 类 号：R114[医药卫生—卫生毒理学]