目的 构建基于HepaRG细胞的三明治培养模型，为评价药物致胆汁淤积风险提供一种高通量且适宜短期研究的参考方法。方法 通过检测5(6)-羧基-2,7二氯荧光素（CDF）及紧密连接蛋白1（ZO-1）荧光强度，结合光学显微镜观察细胞形态，确定模型在96孔培养板中最佳接种密度及建模时间；以对乙酰氨基酚（APAP）为受试物，CCK-8法确定给药浓度；制备三明治培养模型，给药后利用免疫荧光法检测胆汁淤积相关指标，包括CDF、脂质含量、多药耐药蛋白3（MRP3）、法尼醇X受体（FXR）及胆盐输出泵（BSEP），验证模型对评价胆汁淤积的适用性。结果 模型最佳接种密度为每孔7×10⁴个，培养72 h后即可给药。与相同时间的对照组相比，在APAP质量浓度为300～1 200 μg·mL^-1时，细胞脂质含量没有显著性变化；给药1 d，APAP质量浓度在300～600 μg·mL^-1时，CDF荧光强度均显著升高（P＜0.01），给药3 d，CDF荧光强度均显著下降（P＜0.05、0.01）；给药1 d、APAP质量浓度在1 200 μg·mL^-1时，给药3、7 d APAP质量浓度在600～1 200 μg·mL^-1时，MRP3表达量显著下降（P＜0.05、0.01）；给药3、7 d，APAP质量浓度在1 200 μg·mL^-1时，FXR表达量显著下降（P＜0.01）；给药7 d，APAP质量浓度在300 μg·mL^-1时，BSEP表达量显著下降（P＜0.01）。结论 成功建立短期三明治肝细胞培养模型并揭示了APAP导致胆汁淤积的多靶点机制，验证了该模型评价胆汁淤积风险的可靠性。

Objective To develop a sandwich-cultured HepaRG (SCH) model as a high-throughput and short-term research tool for evaluating drug-induced cholestasis risk. Methods The optimal seeding density and modeling time in 96-well plates were determined by detecting the fluorescence intensity of 5(6)-carboxy-2, 7-dichlorofluorescein (CDF) and tight junction protein 1 (ZO-1), and observing the cell morphology under an optical microscope. Acetaminophen (APAP) was used as the test substance, and the CCK8 method was used to determine the drug concentration. Sandwich culture models were prepared, and the bile stasis-related indicators, including CDF, lipid content, multidrug resistance protein 3 (MRP3), farnesoid X receptor (FXR), and bile salt export pump (BSEP), were detected by immunofluorescence after drug administration to verify the applicability of the model for evaluating bile stasis. Results The optimal seeding density was 7×10⁴ cells per well, and the model was ready for drug treatment after 72 h of culture. Under APAP exposure (300—1 200 μg·mL^-1), compared to the lowest concentration, lipid content showed no significant change. After administration for one day, the fluorescence intensity of CDF significantly increased when the APAP mass concentration was 300—600 μg·mL^-1 (P < 0.01). After administration for three days, the fluorescence intensity of CDF significantly decreased (P < 0.05, 0.01). After administration for one day and when the APAP mass concentration was 1 200 μg·mL^-1, and after administration for 3 and 7 days and when the APAP mass concentration was 600—1 200 μg·mL^-1, the expression level of MRP3 significantly decreased (P < 0.05, 0.01). After administration for 3 and 7 days and when the APAP mass concentration was 1 200 μg·mL^-1, the expression level of FXR significantly decreased (P < 0.01). After administration for 7 days and when the APAP mass concentration was 300 μg·mL^-1, the expression level of BSEP significantly decreased (P < 0.01). Conclusion A short-term SCH evaluation model was successfully established, revealing the multi-target mechanism of APAP-induced cholestasis and confirming the model’s reliability for cholestasis risk evaluation.

R285.5

国家重点研发计划“应用纳米材料医疗器械的生物相容性与毒理学研究”资助项目（2022YFC2409702）；药品监管科学全国重点实验室课题“药品杂质遗传毒性评价新技术和生物标志物研究”资助项目（2023SKLDRS0128）