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Research for the in vitro reconstruction of hepatic tissues: an application for an artificial liver.

肝组织体外重建研究：人工肝脏的应用。

基本信息

批准号：
10670213
负责人：
MITAKA Toshihiro
金额：
$ 1.92万
依托单位：
Sapporo Medical University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

We isolated a kind of hepatic progenitor cells, small hepatocytes, from an adult rat liver. A small hepatocyte can clonally proliferate for more than 3 months when the cells are cultured in DMEM supplemented with 10% FBS, 10mM nicotinamide, 1mM ascorbic acid 2-phosphate, 10 ng/ml epidermal growth factor, 10ィイD1-7ィエD1 M dexamenthasone, 0.5 μg/ml insulin, and antibiotics. The cell divides to form a colony and the number of the cells reaches to more than 100 cells within 20 days. Some colonies consist of more than 1000 cells in the culture. Small hepatocytes immunocytochemically show positivity for albumin, transferrin, cytokeratin 8 and 18. In contrast, α-fetoprotein, cytokeratin 14, OC2, and glutathione S-transferase P, which are markers of immature cells, are rarely observed in the early culture period. When small hepatocytes are cultured with hepatic nonparenchymal cells (NPCs) including liver epithelial cells Kupffer cells sinusoidal endothelial cells and stellate cells, piled up cells often appear in the small hepatocyte colonies two to three weeks after plating. In those colonies the invasion of NPCs under the colonies and deposits of extracellular matrix such as type I collagen, type IV collagen, and laminin are observed. The piled up cells show the alteration of cell shape from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin connexin 32 &26, glutamine synthetase, and carbamoylphosphate synthetase are induced. In addition, overlapping NPCs on the piled up cells induced the formation of duct- or cyst-like structures consisting of mature hepatocytes. Thus, small hepatocytes may be "committed progenitor cells" that can further differentiate into mature hepatocytes.

我们从成年大鼠肝脏中分离出一种肝祖细胞--小肝细胞。当细胞在补充有10%FBS、10 mM烟酰胺、1 mM抗坏血酸2-磷酸盐、10 ng/ml表皮生长因子、10 μ g/ml D1-7 μ g/ml D1 M地塞米松、0.5 μg/ml胰岛素和抗生素的DMEM中培养时，小肝细胞可以克隆增殖超过3个月。细胞分裂形成集落，20天内细胞数达到100个以上。有些菌落由培养物中的1000多个细胞组成。小肝细胞免疫细胞化学显示白蛋白、转铁蛋白、细胞角蛋白8和18阳性。相反，在早期培养阶段很少观察到作为未成熟细胞标志物的甲胎蛋白、细胞角蛋白14、OC 2和谷胱甘肽S-转移酶P。当小肝细胞与肝上皮细胞、枯否细胞、窦状内皮细胞和星状细胞等肝非实质细胞（NPC）共同培养时，接种后2 ~ 3周，小肝细胞集落内常出现细胞堆积现象。在这些集落中，观察到集落下NPC的侵入和细胞外基质如I型胶原、IV型胶原和层粘连蛋白的沉积。堆积的细胞显示出细胞形状从相对平坦到立方或矩形的变化。随着细胞结构的改变，白蛋白连接蛋白32和26、谷氨酰胺合成酶和氨甲酰磷酸合成酶的表达被诱导。此外，堆积的细胞上重叠的NPC诱导形成由成熟肝细胞组成的导管或囊肿样结构。因此，小肝细胞可以是“定向祖细胞”，其可以进一步分化成成熟肝细胞。