体外诱导小鼠胚芽干细胞分化为肝细胞的实验研究

目的寻找一种诱导小鼠胚芽干细胞向肝细胞定向分化的最佳诱导剂。方法分离提取孕11 d小鼠胚胎的原始生殖细胞,体外培养扩增后,一部分胚芽干细胞接种到6孔培养板中。向不同孔中分别加入不同浓度的碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）、β-神经生长因子（β-NGF）、维甲酸（RA）、肝细胞提取液进行诱导分化。另一部分胚芽干细胞与胎肝组织共培养。将培养12 d的细胞采用靛青绿摄入试验和白蛋白（ALB）免疫细胞化学染色来鉴定原始生殖细胞向肝细胞分化的情况。结果与胎肝组织共培养和添加了β-NGF、肝细胞提取液的胚芽干细胞能够诱导成肝样细胞,这些肝样细胞能够摄取ICG并表达ALB,上述3组细胞ALB阳性细胞数较对照组明显增多（P〈0.05）。bFGF、EGF、RA并无诱导分化作用。结论β-NGF、胎肝组织产生的细胞因子及肝细胞提取液中的细胞因子可诱导小鼠胚芽干细胞向肝细胞分化。

STUDY ON THE DIFFERENTIATION OF MOUSE EMBRYONIC GERM CELLS INTO HEPATOCYTES IN VITRO

Objective To investigate the condition which can induce embryonic germ cells (EGCs) to differentiate into hepatocytes in vitro, and lay bases for the study of mechanism of EGCs differentiation, so as to seek a new source of seed cells for tissue engineering. Methods The EGCs deriving from gonadal ridges of the mouse embryos on the 11th day were cultured and proliferated in vitro. An aliquot EGCs suspension was cultured in 6-well dishes for 24 hours and then bFGF, EGF, β-NGF, RA and hepatocyte abstraction were added into the dishes respectively for directional differentiation, and another aliquot EGCs was co-cultured with embryonic hepatic cells for differentiation. Morphological differentiation and intake of indiocyanine green (ICG) by EGCs were investigated. The committed differentiation of EGCs into hepatocytes was confirmed by albumin (ALB) immunocytochemistry. Results After addition of β-NGF, hepatocyte abstraction or co-culture with embryonic hepatic cells, the EGCs were differentiated into hepatocyte-like cells which were star-like or oval in shape. These hepatocyte-like cells ingested ICG and expressed ALB immunoreactivity. Conclusion β-NGF, some unknown factors from hepatocyte abstraction and embryonic hepatic cells could induce EGCs differentiating into hepatocytes effectively.