Basic research for the treatments of hepatic diseases by hepatic tissue transplantation

肝组织移植治疗肝病的基础研究

基本信息

批准号：
12670211
负责人：
MITAKA Toshihiro
金额：
$ 2.24万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. We found methods by which SHs could differentiate into mature hepatocytes(MHs) and form three-dimensional structures. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled-up. Although this morphological change occurs when hepatic nonparenchymal cells such as stellate cells invade under SH colonies and produce an extracellular matrix, an overlay of Matrigel^<TM> on SHs can induce similar phenomena. The differentiated SHs express hepatocyte nuclear factor (HNF) 4, HNF6, and CCAAT/enhancer binding protein α, which are expressed in MHs. In addition, not only albumin and transferrin but also carbamoylphosphate synthetase (CPS) I and glutamine synthetase, which are key enzymes of urea synthesis and ammonia metabolism, are also expressed. Furthermore, isozymes of cytochrome P450 can be induced by specific agents. When SH colonies were cultured in a collagen sponge, SHs differentiated into MHs and bile ducts and capillaries were also formed within the scaffold. Now, we are earring out the experiments that the in vitro reconstructed hepatic tissues should be transplant ed to the livers of nude mice or syngeneic rats.SH colonies could be cryopreserved for more than 6 months and the maximum period of the storage was about 90 weeks. About 60 % of SH colonies survive and attached on the dishes. The surviving SHs could proliferate and the average area of SH colonies was about 7.5 times larger at day 15 than at day 1. An amount of albumin secretion into culture medium was about 5 times larger at day 15 than at day 1. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed CPS I and tryptophan 2,3-dioxygenase. The cryopreserved SHs could differentiate into MHs by an overlay of Matrigel.^<TM>

从成年大鼠肝脏中分离出已知是肝祖细胞的小肝细胞（SHS）。我们发现SHS可以区分成熟的肝细胞（MHS）并形成三维结构的方法。 shs shs殖民地有时会从小变为大，从平坦到上升/堆积。尽管这种形态学的变化发生在SH菌落下的肝脏非核细胞（如星状细胞）侵入并产生细胞外基质时，SHS上的Matrigel^<TM>的覆盖物可以诱导相似的现象。分化的SHS表达肝细胞核因子（HNF）4，HNF6和CCAAT/增强子结合蛋白α，在MHS中表达。此外，还表达了蛋白和转铁蛋白和转铁蛋白，而且还表达了尿素合成的关键酶和氨基代谢的关键酶。此外，特定药物可以诱导细胞色素P450的同工酶。当在胶原蛋白赞助商中培养SH菌落时，SHS在支架内也形成了MHS，胆管和毛细血管。现在，我们很早就可以实验，即应将体外重建的肝组织应移植到裸鼠或同性大鼠的生命中。SH菌落可以冷冻保存超过6个月，并且最大的存储期限约为90周。大约60％的SH菌落生存并附着在菜肴上。生存SHS可能会扩散，SH菌落的平均面积比第1天大约7.5倍。在培养基中，将白蛋白分泌量的数量比第1天大约大约5倍。此外，这些细胞还产生了其他血清蛋白（例如转铁蛋白和纤维蛋白），例如纤维蛋白和纤维蛋白，并表达了CPS I和Tearmed CPS I and Phorptppophan 2,3-Dioxygenasase。冷冻保存的shs可以通过矩阵的覆盖层分化为MHS。^<tm>