体外谱系选择法诱导胚胎干细胞神经分化特性的研究

The study on the characters of the neural differentiation from embryonic stem cell induced by lineage selection method

目的研究体外谱系选择法诱导胚胎干细胞(ES)神经分化的特点,建立ES细胞体外神经分化的模型。方法通过悬滴和悬浮培养、Nestin阳性筛选培养、神经样细胞增殖培养以及神经细胞分化培养的方法,建立体外诱导未分化的ES细胞分化为多巴胺能神经元模型;观察各神经分化及各阶段的ES细胞形态,并通过RT-PCR以及荧光免疫化学方法检测各分化阶段ES细胞神经特异表达基因和蛋白的表达特点;通过高效液相检测各阶段分化细胞分泌神经递质多巴胺的能力。结果随着分化培养阶段的延长,分化的ES细胞中可见大量神经样细胞出现,分化末期可见清晰的神经元结构(包括突触);分化早期,仅有部分神经细胞特异基因和蛋白表达,且表达水平低,随着培养阶段延长,特异基因和蛋白均表达,且表达水平明显增加;神经递质多巴胺的水平在未分化和分化早期细胞中未检出,分化的中后期可检测出并呈现随分化时间延长而增加的趋势。结论建立的体外谱系选择培养方法可诱导ES细胞分化获得具有多巴胺能神经元特性的细胞,可为神经发育毒性研究提供模型。

Objective To investigate the characters of nerve differentiation procession induced from mouse embryonic stem cells （ESCs） through lineage selection method and then develop the neural differentiation model in vitro. Methods Dopaminergic neurons development model was induced from undifferentiated ESCs through pendent drop and suspension culture, lineage selection culture, neuron-like cells proliferation and differentiation culture of nerve cells. The morphology was observed and the detection of characteristics of neural specific genes and proteins expression at various differentiation stages was performed by RT-PCR and immunofluorescencc and immunochemistry. The secretion ability of neurotransmitter dopamine at each stages was detected by high performance liquid chromatography （HPLC）. Results With the extending of differentiation time, numerous neuron-like cells were observed in differentiating cells and some neuron structures including synapses were clearly visible in the later differentiation period. In initial differentiation period, only a limited number of neural specific genes and proteins were expressed in low levels while all relating genes and proteins expressed in significantly increased levels with culture proceeding. The neurotransmitter dopamine had not been detected in the undifferentiated and initially differentiating stages but there was a remarkable rising trend in later period. Conclusion The cells acquired from the ESCs differentiation using ITSFn cuhure has the properties of dopaminergic neurons and this is an available model for developmental neurotoxicity research.