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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。
In this study, HepaRG cells were used to establish organoid 3D culture model, and combined with high-content imaging technology to establish an in vitro evaluation method of drug hepatotoxicity. Induction of HepaRG cells into distinct hepatocyte morphology and bile duct-like structures induced by hydrocortisone and dimethyl sulfoxide (DMSO) induces drug-metabolizing enzymes, drug transporters, nuclear receptors, and hepatocyte-specific markers Albumin (ALB) and other related genes to form a stable model of organ-like evaluation of liver function; using high-content imaging technology, specific and multi-target staining by fluorescent dye, from organoid sphere phenotype, The number of viable / dead cells, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) were evaluated for hepatotoxicity. The results showed that the in vitro-like organotypic evaluation model established by HepaRG cells could accurately evaluate the effects of amiodarone (AMD), cyclosporin (CSP) and aspirin (ASP) Toxicity of AMD and CSP both in a concentration-dependent manner led to the total number of organoid spheroids and living cells decreased, and more than 50μmol·L -1 AMD concentration, also led to the rapid death of organoids in the sphere of cells While ASP had no obvious damage to organoids. Both AMD and CSP caused a decrease of MMP and ROS level in a concentration-dependent manner, and the effect intensity of AMD was greater than that of CSP, while ASP had no obvious damage to organoids. In summary, HepaRG cells established high organotypic imaging evaluation method can be used in vitro evaluation of drug hepatotoxicity, and with multi-target high-throughput, the results are intuitive and can be quantified and other advantages.