小鼠胚胎干细胞源性肝细胞的培养体系及分选

Culture system and sorting of hepatocytes derived from mouse embryonic stem cells

背景:已证实细胞可以向肝细胞分化。目前的研究集中在诱导分化上,对分化的肝细胞进行分选和进一步扩增研究较少。目的:比较不同的培养体系对胚胎干细胞源性的肝细胞生长的影响。设计:对比观察。材料:ES-D3细胞由上海细胞生物学研究所丛笑倩教授提供。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18d的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,分别采用Novastem培养体系、肝细胞培养液培养体系和普通培养体系培养3d。前2种培养体系又各分为含去除肝脏小鼠血清、含正常小鼠血清和无血清组3组,普通的培养体系分为含正常小鼠血清和含体积分数为0.1的胎牛血清2组。每组细胞均加入20μg/L肝细胞生长因子、20μg/L表皮生长因子。主要观察指标:细胞计数法分析细胞的增殖率,反转录-聚合酶链反应检测白蛋白的表达以及有限稀释聚合酶链反应法半定量检测胚胎源性肝细胞的含量。结果:①胚胎干细胞源性的肝细胞在不同的培养体系中,生长速度和肝细胞的含量不一样,在加有正常小鼠血清的Novastem培养基中,细胞增殖最快,在无血清的Novastem培养体系中,胚胎干细胞源性的肝细胞含量明显增高。②除了DMEM+体积分数为0.1的胎牛血清组未检测到外,其他组均检测到了白蛋白表达。③在无血清Novastem培养体系中,肝样细胞含量明显增多。结论:无血清的Novastem培养体系能分选和扩增分化的肝细胞,提高肝细胞纯度。

BACKGROUND： Embryonic stem cells can differentiate into hepatocytes. Most studies are on induction and differentiation. A few studies address hepatocyte sorting and further amplification. OBJECTIVE： To compare the effects of various culture systems on hepatocyte growth from embryonic stem cells. DESIGN： A controlled study. MATERIALS： Embryonic stem-D3 cells were supplied by Professor Cong from Shanghai Institute of Cell Biology. METHODS： The embryonic bodies were formed by culturing D3 murine embryonic stem cells in the semi-solid culture medium. 18 days later, embryonic bodies were collected and digested with trypsin. Then these cells were adhered in the dish treated with collagen for 2 hours, cultured with different culture systems, containing Novastem system, cell culture fluid system, and common system for 3 days. The former two culture systems were divided into serum from mice without liver, serum from normal mice and serum-free groups. The common culture system was composed of serum from normal mice group and 0.1 volume fraction fetal bovine serum group. Cells in each group were treated with 20 ug/L hepatocyte growth factor and 20 u g/L epidermal growth factor. MAIN OUTCOME MEASUERS： Cell growth rate was analyzed by cytometry. Albumin expression was measured by using reverse transcription-polymerase chain reaction. Embryo-derived hepatocyte contents were tested utilizing the semi-quantitative limiting dilution polymerase chain reaction. RESULTS： Growth rate and hepatocyte contents were different in embryonic stem cells-derived hepatocytes in various culture systems. In the normal serum Novastem medium, ceils rapidly proliferated. In the serum-free Novastem culture system, contents of hepatocytes derived from embryonic stem cells were significantly increased. Albumin expression was not detected in the DMEM ＋ 0.1 volume fraction fetal bovine serum group, but was measured in other groups. In the serum-free Novastem culture system, hepatocyte contents were significantly increased. CONCLUSION： Novastem without serum can selectively proliferate differentiated hepatocytes, and elevate hepatocyte purity.