建立经优化的促P19鼠胚胎癌细胞神经分化模型

Establishment of optimized neuronal differentiation-promoting model derived from P19 embryonic carcinoma cells

目的:建立P19胚胎癌细胞(embryonal carcinoma cells,EC)定向分化为神经样细胞体外模型,用于快速初步确定小分子化合物促神经发生活性。方法:采用单因素变量法进行培养条件探索,考察全反式维甲酸(all-trans retinoic acid,RA)浓度、悬浮天数、贴壁细胞密度和贴壁后培养液选择对分化的影响。4 d后形成的拟胚体经胰蛋白酶消化为单细胞,并建立分阶段确定评价指标,适用于不同时相评价。光镜观察细胞表型,神经元特异蛋白β-tubulinⅢ免疫荧光染色加以确认。结果:P19细胞悬浮培养4 d,加入RA(终浓度1μmol/L)。以单细胞悬液在1.2×106/ml密度贴壁培养为最佳条件。光镜观察在第8天即有神经元样细胞表型,在第16天可形成神经元样网状结构,两个评价时间点免疫荧光染色均呈β-tubulinⅢ阳性,并与光镜表型相一致。结论:P19细胞分化为神经元样表型,为小分子促神经分化初级筛选提供模型。

Objective： To establish an optimized primary drug screen model of neuronal differentiation using P19 embryonal carcinoma cells. Methods： The final concentration of retinoid acid（RA）,days of suspension culture,manner of adherent culture,suitable cell density and adherent culture medium were tested,respectively.Two stages of neuronal differentiation were examined based on morphological changes and immunocytochemistry analysis of neuronal specific protein β-tubulin Ⅲ. Results： On d 8 of differentiation culture,neuron-like cells were observed with final concentration of 1 μmol/L RA.Neuron-like network was formed on d 16of neuronal differentiation.β-tubulin Ⅲ was positively stained on both stages,indicating P19 cells were differentiated into neurons. Conclusion： The model using RA to induce P19 embryonic carcinoma cells to differentiate into neuron-like cells has been successfully established,which may provide a rapid,phenotypic cell-based platform for primary screening of neurogenesis-promoting drugs.