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a study on isolation and clonal expansion of the stem cell of hepatic stellate cells.

肝星状细胞干细胞的分离和克隆扩增的研究。

基本信息

批准号：
13470001
负责人：
SENOO Haruki
金额：
$ 8.9万
依托单位：
Akita University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470001/
关键词：
stellate cell liver stem cell liver bud septum transversum foregut clone liver diverticulun 肝臓星細胞分化ビタミンA貯蔵細胞間葉系細胞

项目摘要

A stem cell of the hepatic stellate cells was established from mouse embryos. We observed morphologically the process of liver bud formation mixing mesenchymal tissue and foregut in the mouse embryos. During 10-14 somite stage, the hepatic diverticulum was formed and during 18-25 somite stage the foregut entered the septum transversum and the liver bud formation was started. Cell masses obtained from the liver bud containing the hepatic diverticulum and the septum transversum were quickly removed and treated with dispase to get the suspension of isolated cells. The isolated cells were cultured in (1)polystyrene culture dishes, (2)gelatin-coated polystyrene dishes, or (3)polystyrene dishes with a feeder layer of mouse embryo fibroblasts. Three culture media were used : (1)DMEM high glucose medium containing 10% calf serum, (2)DMEM/F12 medium containing 15% calf serum, and (3)ES cell growing medium containing 15% calf serum. To inhibit cell differentiation, a leukemia inhibiting factor (1,000 u/ml) was added to each medium. The best condition was obtaining with ES cell growing medium in gelatin-coated polystyrene dishes. After getting colonies in the dish, each colony was cloned. Cell type was identified by immunofluorescence by using specific antibody against BMP4, AFP, or FLT1(VEGFR), and each antibody demonstrated stellate cells, parenchymal cells, and endothelial cells, respectively. Thus, we established the stem cell of the hepatic stellate cells. The same clone was obtained from flt1 knock out mouse embryos.

从小鼠胚胎中建立了肝星状细胞干细胞。我们从形态学上观察了小鼠胚胎中混合间充质组织和前肠的肝芽形成过程。10-14体时形成肝憩室，18-25体时前肠进入横隔，开始形成肝芽。从含有肝憩室和横隔的肝芽中获得的细胞团被迅速移除并用疾病处理以获得分离细胞的悬液。将分离的细胞分别培养于(1)聚苯乙烯培养皿，(2)明胶包覆聚苯乙烯培养皿，或(3)聚苯乙烯培养皿中，培养皿中有小鼠胚胎成纤维细胞的饲养层。采用三种培养基：(1)含10%牛血清的DMEM高糖培养基，(2)含15%牛血清的DMEM/F12培养基，(3)含15%牛血清的ES细胞生长培养基。为了抑制细胞分化，在每种培养基中加入白血病抑制因子（1,000 u/ml）。以明胶包覆聚苯乙烯培养皿为ES细胞培养基获得最佳条件。在培养皿中获得菌落后，每个菌落都被克隆。使用针对BMP4、AFP或FLT1（VEGFR）的特异性抗体通过免疫荧光鉴定细胞类型，每种抗体分别显示星状细胞、实质细胞和内皮细胞。由此，我们建立了肝星状细胞干细胞。从flt1敲除的小鼠胚胎中获得了相同的克隆。