基于微阵列培养的药物肝毒性评价细胞球模型的建造与模药测评  

Construction of a cell spheroid model for drug hepatotoxicity evaluation based on microarray culture and model drugs assessment

作　　者：陈佳敏 姚香草 苏华龙 陈宗正 魏文博 许重远[1,2] CHEN Jia-min;YAO Xiang-cao;SU Hua-long;CHEN Zong-zheng;WEI Wen-bo;XU Zhong-yuan(Clinical Pharmacy Center,Nanfang Hospital,Southern Medical University,Shenzhen 518035,Guangdong Province,China;National Medical Products Administration Key Laboratory for Research and Evaluation of Drug Metabolism&Guangdong Provincial Key Laboratory of New Drug Screening,School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,Guangdong Province,China;The First Affiliated Hospital of Shenzhen University,Shenzhen518035,Guangdong Province,China)

机构地区：[1]南方医科大学南方医院临床药学中心,广东深圳518035 [2]南方医科大学药学院国家药品监督管理局药物代谢研究与评价重点实验室&广东省新药筛选重点实验室,广东广州510515 [3]深圳大学第一附属医院,广东深圳518035

出　　处：《中国临床药理学杂志》2023年第4期558-562,共5页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金-广东联合基金资助项目(U1401226);南方医院临床研究专项基金资助项目(2019CR015)。

摘　　要：目的 通过微阵列培养,构建由人肝癌HepG2细胞和人永生化肝星状LX-2细胞组成的药物肝毒性评价细胞球模型,采用临床上常用有肝毒性药和保肝药对此模型进行功能性验证和测评。方法 设置不同接种条件,在微阵列中构建由HepG2细胞和LX-2细胞组成的细胞球,通过图像处理软件(Image J)评价细胞球的圆度、致密度和直径,筛选出细胞球的最佳接种条件。用细胞增殖检测试剂盒检测细胞球活性,用荧光定量聚合酶链反应(qPCR)检测细胞球肝功能相关基因表达,用活死细胞双荧光染色及活性检测评价对乙酰氨基酚对细胞球的毒性作用,用谷草转氨酶(GOT)测试盒检测经甘草酸和硫普罗宁处理后的肝损伤细胞球上清液中GOT的含量。结果 细胞球理想接种条件为每微孔500个细胞,细胞比例为HepG2-LX-2=2∶1,圆度为0.84±0.04,致密度为(9.64±0.05)×10^(-1),直径为(175±7)μm,细胞球存活率保持稳定。随培养时间延长,肝特异性标志物白蛋白(ALB)、载脂蛋白(APOB)、谷胱甘肽S转移酶A(GSTA1)mRNA表达升高。细胞球活性随对乙酰氨基酚浓度升高而下降,诱导肝损伤的浓度为20 mmol·L^(-1)。与对照组相比,1 mmol·L^(-1)甘草酸使GOT表达降低55%,10 mmol·L^(-1)硫普罗宁则降低了33%。结论 建立药物肝毒性评价细胞球模型在长期培养中功能保持稳定,与传统模型相比,在评价肝毒性方面具有特定的优势,有助于保肝药物的筛选。Objective To establish a model of drug hepatotoxicity evaluation cell spheroids model consisting of human hepatocellular carcinoma (HepG2) and hepatic stellate cells (LX-2) through microarray culture.The functional validation and evaluation of the model were carried out with hepatotoxicity and hepatoprotective agents commonly used in clinical treatment.Methods Microarray was used for the formation of cell spheroids composed of HepG2 and LX-2 by setting different incubation conditions.Circularity,solidity and diameter were assessed to establish the ideal ratio and size of cell spheroids by image processing software (Image J).The cell viability of the cell spheroids was tested by luminescent cell viability assay.The expression of genes related to liver function of cell spheroids was detected by fluorescence quantitative polymerase chain reaction (qPCR).Immunofluorescence of live/dead staining for cells and cell viability assay were used to assess acetaminophen exposure leading toxicity in cell spheroids.Glutamic-oxaloacetic transaminase (GOT) detection assay kit was used to measure GOT in the supernatant of liver injury cell spheroids after treatment with glycyrrhizic acid and tiopronin.Results The ideal incubation condition of cell spheroids was a combination of HepG2 and LX-2 cells at a2∶1 ratio,500 cells per micropore.It displayed the values of 0.84±0.04 for circularity,(9.64±0.05)×10^(-1)for solidity and (175±7)μm for diameter.Viability of cell spheroids remained stable.And mRNA levels of hepatocyte markers such as albumin(ALB),apolipoprotein(APOB),glutathione S transferase alpha 1(GSTA1) increased with the extention of culture time.Viability of cell spheroids decreased with the increase of acetaminophen concentration.Induced liver injury concentration was determined at 20 mmol·L^(-1).Compared with liver injury control group,1mmol·L^(-1)glycyrrhizic acid and 10 mmol·L^(-1)tiopronin decreased GOT by 55%and 33%,respectively.Conclusion The function of the cell spheroids as a model of drug hepatotoxicity

关 键 词：肝毒性 肝损伤 细胞球 共培养 

分 类 号：R979.1[医药卫生—药品]