目的 構建基于HepaRG細胞的三明治培養(yǎng)模型，為評價藥物致膽汁淤積風險提供一種高通量且適宜短期研究的參考方法。方法 通過檢測5(6)-羧基-2,7二氯熒光素（CDF）及緊密連接蛋白1（ZO-1）熒光強度，結合光學顯微鏡觀察細胞形態(tài)，確定模型在96孔培養(yǎng)板中最佳接種密度及建模時間；以對乙酰氨基酚（APAP）為受試物，CCK-8法確定給藥濃度；制備三明治培養(yǎng)模型，給藥后利用免疫熒光法檢測膽汁淤積相關指標，包括CDF、脂質含量、多藥耐藥蛋白3（MRP3）、法尼醇X受體（FXR）及膽鹽輸出泵（BSEP），驗證模型對評價膽汁淤積的適用性。結果 模型最佳接種密度為每孔7×10⁴個，培養(yǎng)72 h后即可給藥。與相同時間的對照組相比，在APAP質量濃度為300～1 200 μg·mL^-1時，細胞脂質含量沒有顯著性變化；給藥1 d，APAP質量濃度在300～600 μg·mL^-1時，CDF熒光強度均顯著升高（P＜0.01），給藥3 d，CDF熒光強度均顯著下降（P＜0.05、0.01）；給藥1 d、APAP質量濃度在1 200 μg·mL^-1時，給藥3、7 d APAP質量濃度在600～1 200 μg·mL^-1時，MRP3表達量顯著下降（P＜0.05、0.01）；給藥3、7 d，APAP質量濃度在1 200 μg·mL^-1時，F(xiàn)XR表達量顯著下降（P＜0.01）；給藥7 d，APAP質量濃度在300 μg·mL^-1時，BSEP表達量顯著下降（P＜0.01）。結論 成功建立短期三明治肝細胞培養(yǎng)模型并揭示了APAP導致膽汁淤積的多靶點機制，驗證了該模型評價膽汁淤積風險的可靠性。

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

國家重點研發(fā)計劃“應用納米材料醫(yī)療器械的生物相容性與毒理學研究”資助項目（2022YFC2409702）；藥品監(jiān)管科學全國重點實驗室課題“藥品雜質遺傳毒性評價新技術和生物標志物研究”資助項目（2023SKLDRS0128）