鸡ES细胞体外定向诱导分化特性的研究

Research on the Differentiation Characteristics of Chicken Embryonic Stem Cells in vitro

旨在探索鸡胚胎干细胞(ES细胞)体外定向诱导的多向分化潜能。分离鸡X期胚盘细胞,体外培养传代,经鉴定后,采用地塞米松(DEX)、β-甘油磷酸钠(β-GP)和维生素C(VitC)诱导ES细胞向成骨细胞分化;采用维甲酸(RA)、3-异丁基-1-甲基黄嘌呤(IBMX)诱导ES细胞向神经元样细胞分化;采用DEX、胰岛素、IBMX诱导ES细胞向脂肪细胞分化,比较不同浓度组合诱导剂的诱导效果,分别用Von Kossa’s染色法、甲苯胺蓝染色法、免疫组化法、油红O染色法和RT-PCR鉴定诱导产生的细胞。结果显示,ES细胞被诱导3～21d后,分化为成骨细胞,诱导率为40%～72%;被诱导4～7d后,分化为神经元样细胞,诱导率为71%～84%;被诱导2～21d后,分化为脂肪细胞,油红O染色阳性,并表达PPARγ基因。结果表明,在体外条件下,ES细胞可被特异的化学物质定向诱导分化为成骨细胞、神经元样细胞和脂肪细胞,具有多向分化潜能。

The aim of this study was to investigate the differentiation potential of chicken embryonic stem cells（ES cells） under different experimental conditions in vitro.The ES cells isolated from embryos of chicken at the stage X were cultured and subcultured in vitro.After identification,ES cells were induced into osteoblasts in the medium containing desamethason（DEX）,glycerol 2-phosphate disodium salt hydrate（β-GP） and vitamin C（VitC）,and were examined with Immunohistochemical and Von Kossa＇s staining;ES cells were induced into neuron-like cells by retinoic acid（RA） and 3-isobutyl-1-methylxanthine（IBMX）,and were identified by Toluidine Blue and Immunohistochemical staining;ES cells were cultured in DMEM medium plus DEX,insulin and IBMX.The derived cells were determined by Oil Red-O staining,and Peroxisome proliferatoractivated receptor-γ（PPAR-γ） gene transcription was detected by Reverse Transcriptase-polymerase Chain Reaction（RT-PCR）.Osteoblasts were differentiated from ES cells after 3-21 days of induction and with 40%-72% of positive cells efficiency.About 4-7 d after being induced about 71%-84% cells exhibited typical neuron-like cells phenotype.Oil Red-O positive cells formed after 2-21 days of culture in the induction for adipocyte differentiation and conformed by RT-PCR for PPARγ gene transcription.The result indicate that chicken ES cells can differentiate into osteoblasts,neuron-like cells and adipocytes,showing the capability of multipotential differentiation in vitro.