| 林铌,刘鑫磊,孙百阳,俞月,耿兴超,周晓冰,李波.基于3D肝细胞模型评价盐酸胺碘酮重复给药肝毒性[J].中国药事,2022,36(12):1414-1423 |
| 基于3D肝细胞模型评价盐酸胺碘酮重复给药肝毒性 |
| Evaluation of the Hepatotoxicity Induced by Repeated Dosed Amiodarone Hydrochloride Based on 3D Liver Spheroid Model |
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| DOI：10.16153/j.1002-7777.2022.12.011 |
| 中文关键词: 3D肝细胞模型 肝毒性 盐酸胺碘酮 重复给药 利福平 酮康唑 联合用药 |
| 英文关键词: 3D hepatocyte model hepatotoxicity amiodarone hydrochloride repeated administration rifampicin ketoconazole drug combination |
| 基金项目:十三五重大新药创制专项课题“创新药物非临床安全性评价研究关键技术”(编号 2018ZX09201017-001) |
| 作者 | 单位 | | 林铌 | 中国食品药品检定研究院 国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176 | | 刘鑫磊 | 中国食品药品检定研究院 国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176 | | 孙百阳 | 中国食品药品检定研究院 国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176 | | 俞月 | 中国食品药品检定研究院 国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176 | | 耿兴超 | 中国食品药品检定研究院 国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176 | | 周晓冰 | 中国食品药品检定研究院 国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176 | | 李波 | 中国食品药品检定研究院 国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京 100176 |
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| 中文摘要: |
| 目的：建立3D肝细胞微球模型并用于评价盐酸胺碘酮及联用肝药酶诱导剂利福平或抑制剂酮康唑时的重复给药肝毒性。方法：采用诱导分化的HepaRG和HHSteC细胞混合共培养构建3D肝细胞微球模型，对HepaRG诱导分化后形成的胆管结构功能进行验证，活细胞探针标记肝细胞微球中两种细胞并对其分布情况进行检测，免疫荧光染色对肝细胞微球表达的特异性和功能性蛋白进行检验，并对试验周期内模型的肝功能指标稳定性进行连续监测。模型验证后，对每40个肝细胞微球进行连续4或5天的盐酸胺碘酮重复染毒，并联用肝药酶诱导剂利福平或抑制剂胺碘酮进行重复染毒，检测不同给药组的细胞毒性及肝功能指标。结果：本研究构建的3D肝细胞模型可以模拟肝脏胆管结构的外排功能，HepaRG和 HHSteC细胞在微球中以24∶1的比例始终保持较均匀的分布，肝脏特异性和功能性蛋白表达丰富，并能在至少5天内维持肝功能指标稳定。在重复给予胺碘酮时，模型从给药第三天起出现剂量和时间依赖性的细胞毒性作用，且联用利福平(LDH和TBIL升高)或酮康唑(LDH、ALT、ALP和GLU升高)能产生剂量相关的肝毒性协同作用。结论：本研究成功构建更适用于短期重复给药毒性评价的3D肝细胞微球模型，对于体外药物肝毒性筛选和代谢研究具有明显优势，能够进行药物肝毒性标志物的筛选研究。 |
| 英文摘要: |
| Objective: To establish a 3D liver spheroid model to assess the hepatotoxicity induced by repeated dosed amiodarone hydrochloride in combination with liver enzyme inducer rifampicin or inhibitor ketoconazole. Methods: The 3D liver spheroid model was established by mixed co-culture of differentiated HepaRG cells and HHSteC cells, and the function of DMSO-induced bile structure in HepaRG cells was validated. The live cell probes labeled two kinds of cells in hepatocyte spheroids and detected their distribution. The expression of liver-specific and functional proteins were determined by immunofluorescence staining, and the liver function parameters were continuously monitored throughout the period of experiment. Subsequent to the model verifi cation, the repeated administration of amiodarone and its combination with rifampicin or ketoconazole on every 40 liver spheroids was conducted for consecutive 4 or 5 days, and the cytotoxicity and liver function were detected. Results: The 3D liver spheroid model simulated the effl ux function of bile structure, HepaRG cells and HHSteC cells in spheroids were evenly distributed in spheroids at a ratio of 24∶1, the expression of liver-specifi c and functional proteins were abundant, and the liver function remained stable in at least 5 days. When amiodarone was repeatedly dosed, the dose-independent and time-dependent cytotoxicity was observed from day 3 after initial dosage, and its combination with rifampicin (increase in LDH and TBIL) or ketoconazole (increase in LDH, ALT, ALP and GLU) could induce synergistic eff ect on dose-dependent hepatotoxicity. Conclusion: This study has successfully constructed a 3D hepatocyte spheroid model that is more suitable for toxicity evaluation of shortterm repeated administration. Which has obvious superiorty in the in vitro drug hepatotoxicity screening and metabolism studies and could be used to screen drug hepatotoxicity markers. |
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