胎肝干细胞的分离、培养与鉴定

目的体外扩增培养大鼠胎肝干细胞,研究其形态、生物学特性及表面标志物,探讨胎肝干细胞的性质。方法分离培养胎龄12-16d的胎肝细胞,SABC法检测原代、传代后及细胞克隆中的肝干细胞特异表面标志物OV-6、CK-19及nestin的表达。结果原代、传代培养的胎肝细胞部分表达OV-6、CK-19及nestin;培养3d开始出现小细胞团,1个月即形成肉眼可见的细胞集落,5-7d传代一次;细胞克隆几乎全部为干细胞标志阳性细胞。结论胎肝干细胞可通过克隆筛选法进行体外扩增,胎肝内存在nestin阳性干细胞,可能是一种更为原始的干细胞,在胚胎发育中起重要作用。

IN VITRO ISOLATION, CULTIVATION AND IDENTIFICATION OF FETAL HEPATIC STEM CELLS

Objective To study the morphological characteristics,biological properties and phenotypic markers of fetal hepatic stem cells by in vitro culture and proliferation of fetal rat liver.Methods We isolated 12-16 dpc rat fetal hepatic cells and used immunocytochemical method to stain hepatic stem cells expressing protein CK-19,OV-6 and pancreatic progenitor cells marker nestin during the period of primary culture,subculture and cell clone by ABC method.Results Fetal hepatic cells partially expressedCK-19,OV-6 and nestin.Cell groups including 3-5 cells first formed at 3rd day in primary culture and became microscopic cell colonies after one month.Theses cells were subcultured one generation in 5-7 days and almost all expressed the above 3 stem surface markers.Conclusion Fetal hepatic stem cells can be obtained by clone filtration in vitro and have a high proliferation ability.There exist nestin-positive cells in fetal liver which may play an important role as a primordial progenitor cells during embryogenesis and fetation.