胎肝干细胞的分离、培养与鉴定

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

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。

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 ceils partially expressed CK-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.