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.

通过小鼠胚胎建立肝星状细胞的干细胞。我们从形态上观察到肝芽形成的过程混合间充质组织和小鼠胚胎中的前肢。在10-14个节阶段，形成了肝憩室，在18-25节期间，前肢进入了隔膜横向，并开始了肝芽形成。从含有肝憩室和隔膜横向的肝芽中获得的细胞肿块迅速去除并用配件处理，以获取分离的细胞的悬浮液。将分离的细胞培养在（1）聚苯乙烯培养皿中，（2）明胶涂层的聚苯乙烯培养皿，或（3）用小鼠胚胎成纤维细胞的喂食器层的聚苯乙烯培养皿。使用了三种培养基：（1）含有10％小腿血清的DMEM高葡萄糖培养基，（2）含有15％小腿血清的DMEM/F12培养基，以及（3）ES细胞生长含有15％CALF血清的细胞。为了抑制细胞分化，将白血病抑制因子（1,000 U/mL）添加到每个培养基中。最好的条件是在明胶涂层的聚苯乙烯菜肴中使用ES细胞生长培养基获得。在盘中获得殖民地后，每个殖民地都被克隆了。通过使用针对BMP4，AFP或FLT1（VEGFR）的特异性抗体，通过免疫荧光鉴定细胞类型，每种抗体分别显示出星状细胞，实质细胞和内皮细胞。因此，我们确定了肝星状细胞的干细胞。从FLT1敲出小鼠胚胎获得了相同的克隆。