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Establishment of isolation procedure of human hepatic stem cell and tissue engineering technique to reconstitute hepatic tissue using the stem cell.

人肝干细胞分离方法的建立及利用干细胞重建肝组织的组织工程技术。

基本信息

批准号：
11558101
负责人：
SUGIYAMA Toshihiro
金额：
$ 8.7万
依托单位：
Akita Univereity
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

[Aim]ln this study, we first tried to establish the procedure for isolation ofhepatic stem cell from healthy human liver. Next, we examined whether the hepatic stem cell has potential to differentiate into hepatocyte, biliary epithelial cell, or pancreatic cell under tri-dimensional culture condition by using collagen gel developed by Akita Sumitomo Bakelite Co., Ltd. Finally, we aimed the clinical application of topo-organ reconstituted by the ex vivo transplantation of hepatic stem cell.[Achievements](1)We have established a liver epithelial cell line, designated hepatic stem like (HSL) cell, from nonparenchymal fraction of healthy rat liver.(2)The HSL cells are small with 20 μm 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 or 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 how we process the HSL cells to manifest the hepatic function in vivo. To this end, we transplanted the HSL cells which 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. This suggests that the transplanted HSL cells have differentiated into albumin producing cells and function as hepatocytes in vivo. The present study implies the possibility of clinical application of HSL cells.

[目的]建立从健康人肝脏中分离肝干细胞的方法。接下来，我们通过使用由秋田住友电木公司开发的胶原凝胶，在三维培养条件下检查肝干细胞是否具有分化为肝细胞、胆管上皮细胞或胰腺细胞的潜力，最后，对肝干细胞体外移植重建局部器官的临床应用进行了展望。【研究成果】（1）从健康大鼠肝脏非实质部分建立了一株肝上皮细胞系，命名为肝干细胞样（HSL）。（2）HSL细胞体积小，直径约20 μm，核质比高。这些细胞表达甲胎蛋白，但不表达白蛋白和细胞角蛋白19，表明细胞具有未成熟表型。(3)We将HSL细胞在胶原凝胶中培养或与肝星状细胞一起培养以研究其分化潜力。因此，两种培养条件均诱导HSL细胞表达白蛋白，表明HSL细胞能够分化为成熟细胞。（4）最后，我们研究了如何在体内处理HSL细胞以显示肝功能。为此，我们将HSL细胞与胶原凝胶混合或包埋在胶原海绵中移植到无蛋白血症大鼠体内。结果，在移植了HSL细胞的胶原凝胶组和胶原海绵组中，分别有67%和57%的血清白蛋白水平升高的白蛋白缺乏症大鼠。这表明移植的HSL细胞已分化为白蛋白产生细胞，并在体内起肝细胞的作用。本研究为HSL细胞的临床应用提供了可能。