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

小肝细胞(SHS)是从成年大鼠肝脏中分离得到的肝祖细胞。我们发现了SHS分化为成熟肝细胞(MHS)并形成三维结构的方法。群体中的SHS有时会从小到大，从平坦到上升/堆积。尽管当肝非实质细胞(如星状细胞)在SH集落下侵入并产生细胞外基质时会发生这种形态变化，但覆盖在SHS上的Matrigel^&lt；TM&gt；可以引起类似的现象。分化的SHS表达肝细胞核因子4、HNF6和CCAAT/增强子结合蛋白α，它们在MHS中也有表达。此外，不仅表达了白蛋白和转铁蛋白，还表达了尿素合成和氨代谢的关键酶氨基甲酰磷酸合成酶(CPS)I和谷氨酰胺合成酶。此外，细胞色素P450的同工酶可以被特定的药物诱导。当SH集落在胶原海绵中培养时，SHS分化为MHS，支架内还形成胆管和毛细血管。目前，我们正在进行将体外重建的肝组织移植到裸鼠或同基因大鼠肝脏的实验，SH克隆可以冷冻保存6个月以上，最长保存时间为90周左右。大约60%的SH菌落存活并附着在培养皿上。存活的SHS在第15天可以增殖，SH集落平均面积是第1天的7.5倍。第15天分泌的白蛋白大约是第1天的5倍。此外，细胞还产生其他血清蛋白，如转铁蛋白和纤维蛋白原，并表达CPS I和色氨酸2，3-双加氧酶。冷冻保存的SHS可通过Matrigel的覆盖分化为MHS。

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>