目的 構(gòu)建基于HepaRG細(xì)胞的三明治培養(yǎng)模型，為評(píng)價(jià)藥物致膽汁淤積風(fēng)險(xiǎn)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(cè)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(qiáng)度，結(jié)合光學(xué)顯微鏡觀察細(xì)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(shí)間；以對(duì)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(cè)膽汁淤積相關(guān)指標(biāo)，包括CDF、脂質(zhì)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(yàn)證模型對(duì)評(píng)價(jià)膽汁淤積的適用性。結(jié)果 模型最佳接種密度為每孔7×10⁴個(gè)，培養(yǎng)72 h后即可給藥。與相同時(shí)間的對(duì)照組相比，在APAP質(zhì)量濃度為300～1 200 μg·mL^-1時(shí)，細(xì)胞脂質(zhì)含量沒有顯著性變化；給藥1 d，APAP質(zhì)量濃度在300～600 μg·mL^-1時(shí)，CDF熒光強(qiáng)度均顯著升高（P＜0.01），給藥3 d，CDF熒光強(qiáng)度均顯著下降（P＜0.05、0.01）；給藥1 d、APAP質(zhì)量濃度在1 200 μg·mL^-1時(shí)，給藥3、7 d APAP質(zhì)量濃度在600～1 200 μg·mL^-1時(shí)，MRP3表達(dá)量顯著下降（P＜0.05、0.01）；給藥3、7 d，APAP質(zhì)量濃度在1 200 μg·mL^-1時(shí)，F(xiàn)XR表達(dá)量顯著下降（P＜0.01）；給藥7 d，APAP質(zhì)量濃度在300 μg·mL^-1時(shí)，BSEP表達(dá)量顯著下降（P＜0.01）。結(jié)論 成功建立短期三明治肝細(xì)胞培養(yǎng)模型并揭示了APAP導(dǎo)致膽汁淤積的多靶點(diǎn)機(jī)制，驗(yàn)證了該模型評(píng)價(jià)膽汁淤積風(fēng)險(xiǎn)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(guó)家重點(diǎn)研發(fā)計(jì)劃“應(yīng)用納米材料醫(yī)療器械的生物相容性與毒理學(xué)研究”資助項(xiàng)目（2022YFC2409702）；藥品監(jiān)管科學(xué)全國(guó)重點(diǎn)實(shí)驗(yàn)室課題“藥品雜質(zhì)遺傳毒性評(píng)價(jià)新技術(shù)和生物標(biāo)志物研究”資助項(xiàng)目（2023SKLDRS0128）