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背景:胚胎干细胞(ES细胞)是从体外受精胚囊的内细胞团分离建立的、具有发育全能性的细胞系,在特定条件下可向多种细胞分化,是细胞移植治疗很有前途的细胞来源。目的:探明ES细胞是否能被诱导分化为肝细胞,并探索小鼠ES细胞诱导分化为肝细胞的分化条件。方法:在ES细胞培养液中分别加入肝细胞生长因子(HGF)、β-神经细胞生长因子(NGF)和维甲酸(RA)以及与小鼠胎肝细胞共培养,观察分化细胞的形态学变化,逆转录聚合酶链反应(RT-PCR)检测白蛋白和转甲状腺蛋白mRNA水平的表达,免疫组化法检测甲胎蛋白和α1-抗胰蛋白酶蛋白水平的表达。结果:ES细胞培养液中加入HGF和β-NGF 15天后,分化出较多的上皮样细胞,并检测出白蛋白、转甲状腺蛋白mRNA水平的表达和甲胎蛋白、α1-抗胰蛋白酶蛋白水平的表达,表明ES细胞己诱导分化为肝细胞。ES细胞与胎肝细胞共培养2天后,分化出单一形态的上皮细胞,同样有上述肝细胞标志物的阳性表达。RA诱导出的细胞除转甲状腺蛋白mRNA外,无其他肝细胞标志物的阳性表达。结论:在HGF和β-NGF的作用下,有部分小鼠ES细胞被诱导为肝细胞,RA则无此作用。共培养方法也能诱导出肝细胞标志物阳性的细胞,并且细胞形态较单一。小鼠ES细胞有向肝细胞分化的潜能。
BACKGROUND: Embryonic stem cells (ES cells) are cell lines isolated from the inner cell mass of in vitro fertilized embryo sacs and have developmental pluripotency. They can differentiate into many kinds of cells under specific conditions and are promising cells for cell transplantation source. Objective: To investigate whether ES cells can be induced to differentiate into hepatocytes and to explore the differentiation conditions in which mouse ES cells differentiate into hepatocytes. METHODS: Hepatocyte growth factor (HGF), β-nerve growth factor (NGF) and retinoic acid (RA) were added into ES cell culture medium and co-cultured with fetal liver fibroblasts to observe the morphological changes of differentiated cells The mRNA expression of albumin and trans-thyroid protein was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protein expression of alpha-fetoprotein and alpha1-antitrypsin was detected by immunohistochemistry. Results: After 15 days of addition of HGF and β-NGF in ES cell culture medium, more epithelial cells were differentiated and the expression of albumin and transthyretin mRNA and the protein levels of α-fetoprotein and α1-antitrypsin were detected Expression, indicating that ES cells have been induced to differentiate into hepatocytes. After two days of co-culture of ES cells with fetal liver cells, the ES cells differentiated into a single form of epithelial cells with the same positive expression of the above hepatocyte markers. RA-induced cells in addition to the transfer of thyroid protein mRNA, no other positive expression of liver cell markers. Conclusion: Some mouse ES cells were induced to hepatocytes under the action of HGF and β-NGF, while RA did not. Co-culture methods can also induce hepatocyte marker-positive cells, with a single cell morphology. Mouse ES cells have the potential to differentiate into hepatocytes.