摘要
目的基于核酸纳米材料修饰的活细胞技术(nucleic acid nanostructures decorated living cells,NAC)构建肝类器官(liver organoids constructed with NAC,NAC-Liver)模型用于药物肝毒性评估。方法通过核酸纳米折纸材料连接体将原代肝实质细胞、肝窦内皮细胞、库普弗细胞及肝星状细胞以10∶6∶3∶1的比例进行组装,构建具备仿生功能的NAC-Liver模型。采用多重荧光标记技术验证模型中各种细胞的空间分布,并定量分析白蛋白和尿素的分泌水平以评估肝细胞合成功能,进一步通过组织病理学染色评估模型的组织形态及糖脂代谢特征,并检测肝药酶(CYP450家族)活性以分析其代谢功能。根据FDA药物毒性分级选择108种药物进行高通量药物肝毒性评估,系统评价模型对不同药物的毒性响应特征。结果活细胞染色展示了NAC-Liver中不同细胞的分布特征和良好活力。白蛋白和尿素水平检测结果显示,在培养第7、14天,NAC-Liver模型的白蛋白和尿素水平均显著高于2D PHH模型(P值均<0.01)。油红O染色和过碘酸希夫染色图像提示,NAC-Liver模型具有脂质积累和糖原储存能力。在肝药酶活性评估中,NAC-Liver模型在多个关键药物代谢酶(CYP1A2、CYP2B6、CYP2C19、CYP2D6和CYP3A4)催化产生的代谢产物浓度均显著高于2D PHH模型(P值均<0.001)。在高通量药物肝毒性评估中,NAC-Liver模型预测药物毒性的总灵敏度为0.724,能有效区分不同毒性等级的药物。结论NAC-Liver模型为药物肝毒性评价提供了一种创新而有效的方法。
Objective To develop a liver organoid model constructed with nucleic acid nanostructure decorated living cells(NAC-Liver)for the evaluation of drug-induced hepatotoxicity.Methods Primary hepatocytes,hepatic sinusoidal endothelial cells,Kupffer cells,and hepatic stellate cells were assembled in a proportion of 10:6:3:1 using nucleic acid nanomaterial linkers to construct the biomimetic NAC-Liver model.Multifluorescent labeling was employed to validate the spatial distribution of cells within the model.Albumin and urea levels were quantified to assess hepatic synthetic function.Histopathological staining was used to evaluate tissue morphology and glycolipid metabolic features.Cytochrome P450(CYP450)enzyme activity was measured to characterize metabolic function.Based on FDA drug toxicity classifications,108 medications were selected for high-throughput hepatotoxicity assessment of model response to drug toxicity.Results Live-cell staining confirmed distinct spatial distribution and high viability of cells in the NAC-Liver model.Albumin and urea levels in the NAC-Liver model were significantly higher than those in the 2D primary human hepatocyte(PHH)model on day 7 and 14 of culture(all P<0.01).Oil Red O and PAS staining revealed robust lipid accumulation and glycogen storage capacity,respectively.In the assessment of hepatic microsomal enzyme activity,the NAC-Liver model demonstrated significantly higher concentrations of metabolites catalyzed by key drug-metabolizing enzymes(CYP1A2,CYP2B6,CYP2C19,CYP2D6,and CYP3A4)compared to the 2D PHH model(all P<0.01).In high-throughput evaluation of drug hepatotoxicity,NAC-Liver effectively distinguished drugs of varying toxicity levels,with a sensitivity of 0.724 to drug toxicity responses.Conclusion The NAC-Liver model provides an innovative and effective approach for evaluating drug-induced hepatotoxicity.
作者
殷恪帆
王鹤鸣
赵翊丞
宋光启
姚群燕
YIN Kefan;WANG Heming;ZHAO Yicheng;SONG Guangqi;YAO Qunyan(Department of Gastroenterology and Hepatology,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《上海医学》
2025年第6期325-332,共8页
Shanghai Medical Journal
基金
上海市科学技术委员会科技计划项目(22140900700、23ZR1411300)
关键词
类器官
药物性肝损伤
肝毒性
核酸纳米结构
Organoids
Druginduced liver injury
Hepatotoxicity
Nucleic acid nanostructures
作者简介
通信作者:姚群燕,电子邮箱为yao.qunyan@zs-hospital.sh.cn。
