基于类器官3D培养和高内涵成像的药物肝毒性评价模型研究
发布时间：2018-09-05 10:40
【摘要】：本研究采用HepaRG细胞建立类器官(organoids)3D培养模型,并结合高内涵成像技术建立药物肝毒性体外评价方法。通过氢化可的松和二甲基亚砜(DMSO)诱导,使HepaRG细胞分化为明显的肝细胞形态和胆管样结构,并诱导药物代谢酶、药物转运体、核受体及肝细胞特异标志性分子白蛋白(ALB)等相关基因的表达,形成稳定的模拟肝脏功能的类器官体外评价模型;采用高内涵成像技术,通过荧光染料进行特异性、多靶点染色,从类器官球体表型、活/死细胞数量、线粒体膜电位(MMP)和细胞内活性氧(ROS)评价药物肝毒性。结果表明,采用HepaRG细胞建立的类器官体外评价模型可以准确地评价肝毒性阳性对照药胺碘酮(amiodarone,AMD)、环孢霉素(cyclosporin,CSP)及阴性对照药阿司匹林(aspirin,ASP)的毒性差异:其中AMD和CSP均浓度依赖地导致类器官球体的总细胞数和活细胞数下降,并且AMD浓度超过50μmol·L~(-1)时,还导致类器官球体中死细胞数急剧增多,而ASP对类器官无明显损伤作用;AMD和CSP均浓度依赖地导致MMP下降和ROS水平增高,且AMD的作用强度大于CSP,而ASP对类器官无明显损伤作用。综上,HepaRG细胞建立的类器官高内涵成像评价方法可用于药物肝毒性的体外评价,并且具有多靶标高通量、结果直观且可定量等优势。
[Abstract]:In this study, HepaRG cells were used to establish (organoids) 3D culture model and high intension imaging technique was used to establish in vitro evaluation method of drug hepatotoxicity. Induced by hydrocortisone and dimethyl sulfoxide (DMSO), HepaRG cells differentiated into hepatocyte morphology and bile duct like structure, and induced drug metabolizing enzymes and drug transporters. The expression of related genes such as nuclear receptor and hepatocyte specific iconic molecule albumin (ALB) forms a stable in vitro evaluation model of liver function, and uses high intension imaging technology to carry out specific, multi-target staining with fluorescent dyes. Hepatic toxicity was evaluated by organoid globular phenotype, number of living / dead cells, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS). The results show that The in vitro evaluation model established by HepaRG cells can accurately evaluate the toxicity of amiodarone (amiodarone,AMD), cyclosporine (cyclosporin,CSP) and aspirin (aspirin,ASP), in which both AMD and CSP are concentration-dependent on the conductance of hepatotoxic drugs, such as amiodarone (amiodarone,AMD), cyclosporine (cyclosporin,CSP) and aspirin (aspirin,ASP). The total number of cells and the number of living cells in the globules of organs decreased. When the concentration of AMD was more than 50 渭 mol L ~ (-1), the number of dead cells in the organoid spheres increased sharply, while ASP had no obvious damage to the organs. Both ASP and CSP caused the decrease of MMP and the increase of ROS levels in a concentration-dependent manner. The action intensity of AMD was higher than that of CSP, but ASP had no obvious damage to organ like organs. The high intension imaging method developed by HepaRG cells can be used to evaluate the hepatotoxicity of drugs in vitro and has the advantages of multiple targets and high throughput. The results are intuitive and quantifiable.
【作者单位】： 解放军302医院全军中医药研究所;中国医学科学院肿瘤医院;河北北方学院;北京输血医学研究所;
【基金】：国家自然科学基金资助项目(81503350) 北京市自然科学基金资助项目(7152142) 北京市科技新星计划项目(Z16111000490000) 中国博士后科学基金资助项目(2016M590065)
【分类号】：R96
本文编号：2224024
[Abstract]:In this study, HepaRG cells were used to establish (organoids) 3D culture model and high intension imaging technique was used to establish in vitro evaluation method of drug hepatotoxicity. Induced by hydrocortisone and dimethyl sulfoxide (DMSO), HepaRG cells differentiated into hepatocyte morphology and bile duct like structure, and induced drug metabolizing enzymes and drug transporters. The expression of related genes such as nuclear receptor and hepatocyte specific iconic molecule albumin (ALB) forms a stable in vitro evaluation model of liver function, and uses high intension imaging technology to carry out specific, multi-target staining with fluorescent dyes. Hepatic toxicity was evaluated by organoid globular phenotype, number of living / dead cells, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS). The results show that The in vitro evaluation model established by HepaRG cells can accurately evaluate the toxicity of amiodarone (amiodarone,AMD), cyclosporine (cyclosporin,CSP) and aspirin (aspirin,ASP), in which both AMD and CSP are concentration-dependent on the conductance of hepatotoxic drugs, such as amiodarone (amiodarone,AMD), cyclosporine (cyclosporin,CSP) and aspirin (aspirin,ASP). The total number of cells and the number of living cells in the globules of organs decreased. When the concentration of AMD was more than 50 渭 mol L ~ (-1), the number of dead cells in the organoid spheres increased sharply, while ASP had no obvious damage to the organs. Both ASP and CSP caused the decrease of MMP and the increase of ROS levels in a concentration-dependent manner. The action intensity of AMD was higher than that of CSP, but ASP had no obvious damage to organ like organs. The high intension imaging method developed by HepaRG cells can be used to evaluate the hepatotoxicity of drugs in vitro and has the advantages of multiple targets and high throughput. The results are intuitive and quantifiable.
【作者单位】： 解放军302医院全军中医药研究所;中国医学科学院肿瘤医院;河北北方学院;北京输血医学研究所;
【基金】：国家自然科学基金资助项目(81503350) 北京市自然科学基金资助项目(7152142) 北京市科技新星计划项目(Z16111000490000) 中国博士后科学基金资助项目(2016M590065)
【分类号】：R96
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