Development of a hybrid artificial liver using small hepatocytes with high proliferative activity

本项目旨在为开发具有高增殖活性的小肝细胞混合人工肝和/或肝细胞移植的肝支持疗法进行基础研究。(1)在评估小肝细胞增殖活性和小肝细胞在切除70%肝脏后肝再生中的作用的研究中，小肝细胞在2周内表现出高增殖活性，DNA量增加4倍，肝细胞核倍性分析显示小肝细胞在大鼠模型中对成年肝细胞的缺乏起到了补足作用。(2)在使用前寻找合适的肝细胞培养环境的研究中，研究了肝细胞在蜂窝状可生物降解多孔膜上的培养和小肝细胞球体的形成。孔径减小（~ 10μm）和部分水解可增强肝细胞的扩散程度，更多的膜表面可促进肝细胞的迁移和球形形成。在67rpm的振动培养中，小肝细胞在3小时内开始聚集，并在6小时内成为直径约50 ~ 80μm的球体。球体的形状在培养中保存了10天。(3)在小肝细胞经门静脉移植到白蛋白血症大鼠体内的研究中，血清白蛋白在四周内升高2.5倍。移植肝免疫组织染色可见大量白蛋白阳性细胞。小肝细胞被认为是一种很有前途的细胞移植和杂交人工肝的候选细胞。(4)小肝细胞具有较高的增殖活性，但不是永生化细胞。因此，将基因诱导作为小肝细胞永生化的基础研究。在单个胰岛中发现了一种有效的基因诱导方法，其诱导率约为100%。结果表明，小肝细胞作为混合人工肝和/或肝细胞移植是肝支持治疗的良好细胞来源。少

This project was aimed to make a fundamental investigation for developing hepatic support therapies as a hybrid artificial liver and/or hepatocyte transplantation using small hepatocytes with high activities of proliferation.(1)In the study for evaluating proliferative activity of small hepatocytes and the role of small hepatocytes in hepatic regeneration after 70% resection of the liver, small hepatocytes showed high proliferative activity with an increase in DNA amount four times in two weeks and an analysis of nuclear ploidy of hepatocyte showed small hepatocytes play a supplying role for a deficiency of adult hepatocytes in rat model.(2)In the studies for the investigation for suitable environment for hepatocyte culture before use, hepatocyte culture on a honeycomb patterned biodegradable porous film and formation of small hepatocyte spheroid were investigated. The degree of spreading of hepatocyte was enhanced with decrease of the pore size (〜10μm) and partial hydrolysis of the fi … More lm surface enhanced the migration and spheroid formation of the cultured hepatocyte. In a shaking culture of 67rpm, small hepatocytes start to aggregate in three hours and became spheroids approximate 50〜80μm in diameter in six hours. Shape of the spheroics has been kept for 10 days in culture.(3)In the study for small hepatocyte transplantation into analbuminemia rat through portal vein, serum albumin elevated 2.5 times in four weeks. Massive albumin positive cells were observed in the immunohistological staining of the transplanted liver. Small hepatocytes are considered as a promising candidate cell for cell transplantation and for a hybrid artificial liver.(4)Small hepatocytes possess high proliferative activity, but they are not immortalized cells. Therefore gene induction was investigates as a basic study for immortalization of small hepatocytes. An effective method of gene induction as of around 100% was found in singularized islets.It was concluded that small hepatocytes were suggested to be a good cell source of hepatic support therapies as a hybrid artificial liver and/or hepatocyte transplantation. Less