胚胎干细胞向神经细胞诱导分化的实验研究

Differentiation of embryonic stem cells into neuronal cells in vitro

目的 探讨胚胎干细胞 (ES)向神经细胞定向分化发育的可能性。方法 ES D3在无小鼠白血病抑制因子 (mLIF)存在的条件下培养ES细胞 4d ,形成ES集落 (即胚胎样小体 ) ,经胰酶消化打散后种植于粘附性培养皿上 ,再经全反视黄酸 (RA)诱导 4d。然后分别作免疫组织化学、RT PCR检测 ,鉴定分化细胞的特征。结果 上述细胞表达MAP 2和GFAP蛋白。RT PCR分析进一步证明这些细胞能表达γ 氨基丁酸 (GABA)γ亚单位和酪氨酸羟化酶以及脑因子 1神经相关基因。结论 ES细胞在体外能有效地被诱导成具有多种神经元特性的神经样细胞 ,可为神经移植提供源源不断的材料。

Objective To investigate the probabilities of embryonic stem (ES) cells differentiating into neuronal cells in vitro. Methods Mouse ES-D3 cells were cultured on the ES conditioned media without mLIF (mouse leukemic inhibitory factor, mLIF) for 4 days until ES cell aggregations was formed (embryonic bodies,EBs). The EBs were dissociated with trypsin and exposed to retinoic acid (RA) for another 4 days, and then replated onto adhesive substrate. Immunohistochemistry and RT-PCR assay were performed to identify the properties of the differentiated cells. Results The treated cells expressed neuronal proteins such as MAP-2 and GFAP. RT-PCR assay further identified that the induced cells expressed transcripts for neural associated genes including GABA-γ subunit receptor, brain factor-1 and tyrosine hydroxylase (TH). Conclusion ES cells can be successfully induced into neuronal cells expressing multiple properties of neurons in vitro, which could provide resourceful materials for neural transplantation.