Establishment of cell transplantation therapy for Wilson's disease using hepatic stem cells.

[目的]在本研究中，我们旨在利用先前从大鼠肝脏中分离的肝干细胞建立代谢性肝病的细胞移植治疗。为此，我们检测了移植的肝干细胞在肝脏中的功能，细胞移植是否有效治疗包括威尔逊病在内的代谢性肝脏疾病。【成果】(1)利用健康大鼠肝脏非实质部分建立肝上皮细胞系，命名为肝干样细胞（HSL）。(2) HSL细胞体积小，直径约20μm，核质比高。这些细胞表达α-胎蛋白，但不表达白蛋白和细胞角蛋白19，表明细胞具有不成熟表型。(3)将HSL细胞与肝星状细胞在胶原凝胶中培养，观察其分化能力。因此，两种培养条件均诱导HSL细胞表达白蛋白，表明HSL细胞能够向成熟细胞分化。(4)最后，我们检测了HSL细胞移植后能否在体内表现出肝功能。为此，我们将HSL细胞与胶原凝胶混合或包埋于胶原海绵中移植到无抗体大鼠体内。结果，无抗体血症大鼠血清白蛋白水平在胶原凝胶移植的大鼠中升高67%，在胶原海绵移植的大鼠中升高57%。在这些大鼠中，血清白蛋白水平在移植后10周升高，并维持到移植后25周。提示HSL细胞具有分化潜能，移植后的HSL细胞在体内可分化为产生白蛋白的细胞并具有肝细胞功能。本研究提示HSL细胞在代谢性肝病的临床应用的可能性。

[Aim]In this study, we aimed the establishment of cell transplantation therapy for metabolic liver diseases using hepatic stem cells, previously isolated from a rat liver. To this end, we examined function of the transplanted hepatic stem cells into liver whether cell transplantation was effective to treat metabolic liver diseases including Wilson's disease.[Achievements](1) We have established a liver epithelia cell line, designated hepatic stem like (HSL) cell, from nonparenchymal fraction of healthy rat liver. (2) The HSL cells are small with 20μm in diameter and showed high nucleus/cytoplasm ratios. These cells expressed α-fetoprotein, but not albumin nor cytokeratin 19, indicating the cells have immature phenotype. (3) We cultured the HSL cells in collagen gels cr with hepatic stellate cells to investigate their potential to differentiate. Consequently, both of culture conditions induced the HSL cells to express albumin, showing that the HSL cells are able to differentiate into mature cells. (4) Finally, we examined whether the HSL cells will be able to manifest hepatic function in vivo after transplantation. To this end, we transplanted the HSL cells that were mixed with collagen gel or embedded in collagen sponge into analbuminemic rats. As a result, analbuminemic rats that showed the increased levels of serum albumin were 67% in the rats transplanted the HSL cells with collagen gel and 57% in the rats with collagen sponge. In those rats, the levels of serum albumin increased ten weeks after transplantation and maintained until 25 weeks after transplantation. These results suggest that the HSL cells are potential to differentiate and that the transplanted HSL cells differentiated into albumin producing cells and function as hepatocytes in vivo. The present study implies the possibility of clinical application of HSL cells for metabolic liver diseases.