选择性扩增小鼠胚胎干细胞源性的肝细胞

SELECTIVE PROLIFERATION OF HEPATOCYTES FROM MURINE EMBRYONIC STEM CELLS IN VITRO

目的探讨胚胎干细胞源性的肝细胞体外培养扩增条件。方法采用半固体培养D3小鼠胚胎干细胞系,形成胚胎体。将来源于18 d胚胎体用胰酶消化为单个细胞,在胶原处理过的培养皿中贴壁2h,加入含有肝细胞生长因子和上皮细胞生长因子的肝细胞生长液培养,3 d后,荧光免疫染色检测白蛋白(ALB)的表达,RT-PCR检测白蛋白以及其他肝细胞的特异性蛋白的mRNA表达。结果胚胎干细胞源性的肝细胞在无血清的HGM+HGF+EGF中可以生长,荧光免疫染色显示大约30%～50%的细胞表达ALB,RT-PCR显示它表达ALB和其他肝细胞的特异性蛋白的mRNA。结论 HGM+HGF+EGF可以支持来源于胚胎体的肝细胞的生长和增殖。

Objective To optimized the culture condition of hepatocytes from murine embryonic stem （ES） ceils. Methods The embryonic bodies were formed by culturing D3 murine ES cells in the semi - solid culture medi-um, 18 day embryonic bodies were digested with trypsin and attached on collagen coated dishes for 2 hours. The cells were cultured in the Hepatocyte Growth Medium （HGM） containing Hepatocyte Growth Factor （HGF） and Epidermal Growth Factor （EGF）. Immunofluorescente staining and RT - PCR were performed to analyze the expression of alumin （ALB） protein and mRNA level of albumin （ALB） and other hepatocyte specific genes. Results Hepatocytes from Murine Embryonic Stem Cells can grow and proliferate in the culture system of HGM ＋ EGF ＋ HGF without serum. Conclusion HGM ＋ EGF ＋ HGF can selectively proliferate Hepatocytes from Murine Embryonic Stem Cells.