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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 采用细胞因子四步诱导分化方法,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23d,将hiPS细胞分化成类肝细胞。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P450 3A4酶(CYP3A4)和α1-抗胰蛋白酶(AAT)等蛋白的表达;采用qPCR法检测肝细胞特异性基因: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(相当于生药量1,2,4,8 mg·mL~(-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排泌功能。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,可以做为中药肝毒性筛选的模型细胞。
Objective To explore the feasibility of establishing hepatic toxicity model induced by human induced pluripotent stem cells (hiPS cells) and to establish hepatic toxicity test model for in vitro toxicity evaluation of Chinese medicine. Methods Four-step induction of differentiation by cytokines was performed by adding Activin A, bone morphogenetic protein-4 (BMP-4), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor ( On the other hand, cytokines such as HGF and Oncostatin M were continuously cultured for 23 days to differentiate hiPS cells into hepatocyte-like cells. The cell-specific immunofluorescence method was used to detect the specific markers of hiPS cells in different stages of hepatocyte differentiation: forkhead box transcription factor A2 (FOXA2), sex determination region Y box protein 17 (SOX17), alpha-fetoprotein (AFP), and white Expression of protein (ALB), hepatocyte cytochrome P450 3A4 enzyme (CYP3A4) and α1-antitrypsin (AAT); detection of hepatocyte-specific genes by qPCR: alpha fetoprotein (AFP), albumin (ALB) , Cytokeratin 8 (CK8), cytokeratin 18 (CK18) and cytokeratin 19 (CK19) gene expression; using indocyanine green (ICG) uptake test to detect excretion of hepatocytes. The hepatotoxicity test was performed on hepatocytes using Tripterygium lyophilis powder (equivalent to 1,2,4,8 mg·mL -1 crude drug). After 24 hours, Aspartate aminotransferase (AST) and alanine aminotransferase were tested. (ALT), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) content or activity. Results FOXA2 and SOX17 proteins were detected at the induction stage of endoderm. AFP and ALB were expressed at the stage of hepatocyte differentiation and expansion. CYP3A4 and AAT were expressed at the stage of hepatocyte maturation; hepatocyte-specific genes AFP, CK19 and ALB CK8 and CK18 were all expressed in hepatocyte-like cells. There was significant difference between before and after induction (P<0.05). ICG uptake experiments showed that hepatocyte-like cells had specific excretion of hepatocytes. Compared with the control group, the activities of ALT and AST were significantly increased, the MDA concentration was significantly increased, and the activity of GSH and SOD were significantly decreased in each dose group of Tripterygium wilfordii (P<0.05, P<0.01). Conclusion The method of inducing hepatocyte formation by hiPS cells is feasible. The induced hepatocytes have the function of normal human hepatocytes and can be used as a model cell for hepatic toxicity screening of traditional Chinese medicine.