用单层分化法进行胚胎干细胞神经分化的研究

A Study on Neural Differentiation from Mouse Embryonic Stem Cells by Monolayer Differentiation

用单层分化法研究了 ES细胞从 0～ 316 h(第 14天 )的神经分化过程。无饲养层培养的 ES细胞在无血清的N2 B2 7培养基中开始神经分化。从 72 h开始 ,神经外胚层的特异性转录因子 Sox1表达 ,在克隆的边缘逐渐出现向外迁移的细胞 ,形状呈圆形或椭圆形 ,有 1～ 2个细胞突起。在 191h(第 8天 )之后 ,细胞可迁移至克隆边缘的 2 0 0 μm之外 ,这些有细长突起的细胞表达神经元特异性蛋白 Tau。以后 ,迁移细胞的突起开始交叠成网状。 316 h(第 14天 )时 ,细长的纤维可跨越在不同的克隆之间 ,长达上千微米。此时 ,免疫染色的结果表明 ,在克隆的中央有较多的圆形 Nestin阳性细胞 ,而克隆的边缘有大量的 β- tubulin 阳性细胞 ,它们交织成复杂的网状图像。阶段性表达基因的表达时序研究表明 ,ES细胞的特异性转录因子 Oct4开始减弱时 ,Sox1表达开始 ,此时细胞由全能状态进入早期神经分化状态 ;Tau的表达是 Sox1表达的下游事件。这一时序与胚胎发育中神经发生的时序相符。单层分化法能直观地展示 ES细胞的神经分化过程 ,可作为研究细胞分化。

It would be of enormous benefit if there are effective systems to produce neural cells for the replacement of cell lost as a result of injuries of various neurological diseases.Given the complexity of neural development,the mechanism of neuroectoderm formation from pluripotent founder cells is questionable.By monolayer differentiation,an adherent monoculture without multicellular aggregation or induction by retinoid acid,we investigate neural differentiation from mouse embryonic stemcells.On 96 h after changing media with N2B27,differentiated cells moved away from the edges of ES cell clones.On 191 h(D8),they could move as tar as 200 μm from the edges.On 316 h(D14),many fibers formed the complex network,and some connected different clones (1 000) μm apart.These cells were β-tubulin Ⅲ positive whereas the cells in the middle of the clone were still nestin positive.The timing of genes expression,such as Oct4,Sox1 and Tau,was corresponded the sequence which is showed in embryonic neural genesis.The network suggests that these differentiated cells are of potency to from complex connection.