通过hiPS细胞诱导生成类肝细胞建立肝毒性检测模型及其初步评价  

Establishment and Preliminary Evaluation of Hepatotoxicity Testing Model Generated by hiPS Cells-derived Hepatocytes

作　　者：胡秋艳 谭庆龙 徐雯星 刘春萍 曾星 HU Qiuyan;TAN Qinglong;XU Wenxing;LIU Chunping;ZENG Xing(The Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510006 Guangdong,China)

机构地区：[1]广州中医药大学第二附属医院,广东广州510006

出　　处：《中药新药与临床药理》2018年第1期91-96,共6页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：广东省自然科学基金项目(2014A030313415);广东省中医药科学院科学技术研究专项(YK2013B2N10)

摘　　要：目的探讨通过人诱导多能干细胞(human induced pluripotent stem cells,hiPS cells)诱导生成类肝细胞,建立肝毒性检测模型并用于中药毒性体外评价的可行性。方法采用细胞因子4步诱导分化方法,通过加入激活素A(Activin A)、骨形态发生蛋白-4(BMP-4)、成纤维细胞生长因子-2(FGF-2)、肝细胞生长因子(HGF)、制瘤素M(Oncostatin M)等细胞因子,连续培养23 d,将hiPS细胞诱导分化成类肝细胞。采用细胞免疫荧光法检测hiPS细胞向类肝细胞分化过程不同阶段的特异性标记:叉头框转录因子A2(FOXA2)、性别决定区Y框蛋白17(SOX17)、甲胎蛋白(AFP)、白蛋白(ALB)、肝细胞色素P4503A4酶(CYP3A4)和α1-抗胰蛋白酶(AAT)等蛋白的表达;采用q PCR法检测类肝细胞特异性基因:甲胎蛋白(AFP)、白蛋白(ALB)、细胞角蛋白8(CK8)、细胞角蛋白18(CK18)和细胞角蛋白19(CK19)的基因表达;采用吲哚青绿(ICG)摄取实验检测类肝细胞排泌功能。采用雷公藤冻干粉(1,2,4,8 mg·m L^(-1))对类肝细胞进行肝毒性实验,作用24 h后检测谷草转氨酶(AST)、谷丙转氨酶(ALT)、丙二醛(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)的含量或活性。结果检测到FOXA2、SOX17蛋白在内胚层诱导阶段有表达,AFP、ALB在肝细胞分化与扩增阶段有表达,CYP3A4、AAT在肝细胞成熟阶段有表达;肝细胞特异性基因AFP、CK19、ALB、CK8、CK18在类肝细胞中均有表达,诱导前后比较差异有统计学意义(P<0.05);ICG摄取实验显示类肝细胞具有肝细胞的特异性ICG摄取功能。与对照组比较,雷公藤各剂量组的ALT及AST活性显著升高、MDA浓度显著升高、GSH及SOD活性显著降低,差异均有统计学意义(P<0.05,P<0.01)。结论通过hiPS细胞诱导生成类肝细胞的方法具有可行性,诱导得到的类肝细胞具有人正常肝细胞的功能,可以作为中药肝毒性筛选的模型细胞。Objective To explore the feasibility of traditional Chinese medicine toxicity evaluation in vitro through establishing hepatotoxicity test model of human induced pluripotent stem(hiPS)cells-derived hepatocytes.Methods The generation of hiPS cells-derived hepatocytes was carried out through a four-step protocol included addition of the Activin A,bone morphogenetic proteins-4(BMP-4),fibroblast growth factor-2(FGF-2),hepatocyte growth factor(HGF)and Oncostatin M for 23 days to induce differentiation.The endoderm markers Forkhead-box A2(FOXA2)and SRY-box containing gene 17(SOX17),the hepatic cell markers alphafeto protein(AFP)and albumin(ALB),hepatic cell function markers cytochrome P4503A4(CYP3A4)and alpha 1-antitrypsin(AAT)were detected by immunofluorescence method;the expression of hepatic cell gene AFP,ALB,cytokeratin 8(CK8),cytokeratin 18(CK18)and cytokeratin 19(CK19)were measured by quantitative PCR(q PCR);the hepatic cell uptake function was detected by indocyanine green(ICG)uptake test.Hepatotoxicity test was performed by adding the Tripterygium lyophilized powder to the hiPS cells-derived hepatocytes(with the concentrations of 1,2,4,8 mg·mL^-1 which equal to crude drug)for 24 hours.We detected the activity or concentration of Aspartate transaminase(AST),Alanine aminotransferase(ALT),Malondialdehyde(MDA),Glutathione(GSH)and Superoxyde Dismutase(SOD).Results After 23 days’induction,the results of immunofluorescence showed that the endoderm markers FOXA2 and SOX17were expressed at stage of endoderm induction,the hepatic cell markers AFP and ALB were expressed at stage of hepatic specification and hepatoblast expansion,and hepatic cell function markers AAT and CYP3A4 were expressed at stage of hepatic maturation.The hepatic cell gene AFP,ALB,CK8,CK18 and CK19 were all expressed in the hiPS cells-derived hepatocytes.The result of ICG uptake test indicated that the induced cells had the function of hepatic cells.Compared with the control,the activities of ALT,AST and MDA,and the concentration of GSH significant

关 键 词：人诱导多能干细胞 类肝细胞 细胞免疫荧光法 实时荧光定量聚合酶链式反应 吲哚青绿 雷公藤 肝毒性 

分 类 号：R285.5[医药卫生—中药学]