人源iPSC的肝细胞分化及其三维体系中代谢功能研究

Hepatocyte model has been developed as one of the most widely used models for the studies of drug metabolism. However, due to the limitation of the existing hepatocyte metabolism models, that is, human hepatocytes are a scarce and expensive resource which functions only short-term in vitro, this project therefore aims at generating hepatocyte-like cells from human induced pluripotent stem cells (iPSCs) through induced differentiation. The generated hepatocyte-like cells will then be cultured in three-dimensional scaffolds to construct the three-dimensional culture model for human iPSCs derived-hepatocytes, which is expected to overcome the above-mentioned limitation. The contents include the following: non-viral nanoparticles-mediated generation of human iPSCs; induced directional differentiation of human iPSCs into hepatocytes; investigation about the expression of drug metabolic enzymes of the two-dimensional and three-dimensional cultured human iPSCs derived-hepatocytes and cell viability; and study on the metabolism of the bioactive ingredients from the Traditional Chinese Medicine in human iPSCs derived-hepatocytes. In addition, comparisons between the human iPSCs derived-hepatocytes and normal hepatocytes will be conducted via evaluation of the function and metabolic activity of these two types of hepatocytes, followed by detection of the expression changes in the drug metabolic enzymes. Furthermore, the duration of the cell viability of the human iPSCs derived-hepatocytes in the three-dimensional culture model will also be investigated. Following this development, the metabolism of the bioactive ingredients from the Traditional Chinese Medicine in this new model will be investigated through the observation of their toxicity to the human iPSCs derived-hepatocytes and induction/inhibition effects on the common drug metabolic enzymes, as well as the analysis of their possible metabolic products. In conclusion, the objective of this project is to construct a novel three-dimensional metabolic model for human iPSCs-derived hepatocytes, thus providing new thoughts and new technology for the study of drug metabolism.

肝细胞模型已成为药物代谢研究中应用最广泛的模型之一。本项目将人源多能干细胞诱导分化为肝样细胞后种植于三维支架中，构建人源化肝细胞的三维培养模式，以期克服现有肝细胞代谢模型的不足：人的肝细胞来源困难、体外活性维持时间短。内容包括：非病毒纳米粒介导的人源诱导多能干细胞研究、人源iPSC向肝细胞的定向诱导分化、二维培养药物代谢酶表达及细胞活性研究、三维培养药物代谢酶表达及活性持续时间研究、中药活性成分在人源化肝细胞中的代谢研究。通过评价肝细胞功能和代谢活性、检测药物代谢酶的表达变化，比较源自iPSC的肝细胞和正常肝细胞的异同，同时考察三维培养体系中肝细胞活性的维持时间。研究中药活性成分在新模型中的代谢及对诱导肝细胞的损伤及药物代谢酶的调控作用，分析其可能的代谢产物。本项目旨在构建人源肝细胞三维代谢新模型，从而为药物代谢研究提供新思路、新技术。

本课题重点围绕人源多能干细胞诱导分化为肝样细胞，而后种植于三维支架中，构建人源化肝细胞三维培养模式开展研究，内容包括：1、制备、表征磷酸钙-OSKM非病毒纳米粒，并进行人源诱导多能干细胞（iPSCs）研究，成功获得胚胎干细胞样、阳性表达碱性磷酸酶和各种多能干细胞标记物（包括OCT4, KLF4, NANOG, TRA-81）的人源iPSCs；2、人源iPSCs向肝细胞定向诱导分化。运用免疫荧光染色和ELISA法检测肝细胞特异性蛋白表达水平，结果表明：人源iPSCs诱导的肝细胞阳性表达特异性蛋白ALB、AFP、CK18，对天然产物杠柳苷元在大鼠肝微粒体中的代谢进行初步研究，为后续人源化肝细胞代谢研究奠定基础；3、二维培养体系中人源肝细胞药物代谢酶表达及细胞活性研究。显微镜观察人源诱导肝细胞呈双核或多核形态；Western blot测得人源诱导肝细胞药物代谢酶（CYP450和GST）的表达与正常肝细胞相当；试剂盒测得人源诱导肝细胞中各种酶（谷丙转氨酶、谷草转氨酶、乳酸脱氢酶）的表达、白蛋白水平、尿素质量浓度以及消耗葡萄糖量均与人正常肝细胞趋于一致，说明人源诱导肝细胞具有类似于人正常肝细胞的功能；4、制备并优化组织工程三维支架，考察三维培养体系中人源肝细胞药物代谢酶表达及活性持续时间。与二维体系相比，三维体系利于人源肝细胞的生长和增殖，HE染色和免疫组化染色结果表明：人源诱导肝细胞在三维体系中形成双核或多核细胞的肝样组织，阳性表达肝细胞极化蛋白BSEP，药物代谢酶的表达和活性均有明显增加，与人正常肝细胞基本一致，并具有与人正常肝细胞相当的药物代谢活性；5、对中药活性成分在人源化肝细胞中的代谢进行研究。与二维体系相比，三维体系中药物对肝细胞的毒性明显减小；采用RT-PCR、Western blot、LC-MASS等考察西利宾胺、黄芩苷等对药物代谢酶表达的调控和代谢活性的影响，结果表明：西利宾胺将诱导人源肝细胞代谢后产生了两个明显的代谢物峰，通过LC-MASS和查证文献，其中分子量为657.1474 Da的代谢峰结构确证为水飞蓟宾糖醛酸化产物（silybin B-7-O-β-glucuronide），分子量为655.1474 Da的代谢峰是首次发现的水飞蓟宾代谢产物，推测为水飞蓟宾母核糖醛酸化物。本项目成功构建了人源肝细胞三维代谢新模型，为药物代谢研究提供了新思路、新技术。