小鼠胚胎干细胞分化扩增期连续低密度传代对原始细胞中Oct-4阳性细胞比例及神经分化能力的影响

Impact of Serially Low Density Passage on Oct-4 Positive Percentage and Neurogenesis Potential of Mouse ES Derived Progenitors in Differentiated Expanding Stage

目的:探讨在分化扩增期采用连续低密度传代的方法是否能降低小鼠胚胎干细胞向神经细胞分化的前体细胞中Oct-4阳性细胞的比例,以及对神经分化能力的影响。方法:采用"五步法"将小鼠胚胎干细胞向神经元分化,进入扩增期后采用连续低密度传代的方法连续传10代。然后应用细胞免疫组化鉴定Oct-4阳性细胞、神经元与胶质细胞、流式细胞仪检测Oct-4阳性细胞比例、凋亡试剂盒测定细胞凋亡。结果:流式细胞仪检测出扩增期连续低密度传代得到的前体细胞中Oct-4阳性细胞的比例由16.17±4.8%降至4.33±1.63%,扩增期低密度传代细胞和正常高密度传代细胞的细胞凋亡率鉴定分别为15.16±3.64%和11.88±2.63%,步骤5诱导分化后的细胞GFAP和Tuj-1免疫组化染色呈阴性。结论:低密度传代培养可以减少分化后Oct-4阳性细胞的比例,且该比例下降不是由Oct-4阳性细胞的凋亡引起的。同时可能是因为低密度传代培养和高密度相比引起了细胞的质变、或者改变了前体细胞向神经元分化的某种微环境,导致了前体细胞不能分化为神经细胞。提示高密度培养在前体细胞向神经元分化过程中具有重要作用,高密度和低密度培养的比较,提供了研究ES细胞向神经元分化机制的新平台和研究方向。

Objective： To identify whether serially low density passage could decrease the Oct-4 positive cell percentage and maintain the ability of neurogenesis of mouse ES derived progenitors in expanding stage. Methods： Mouse ES were differentiated into neurons according to five-step method and given more than 10 continuous passage by low seeding density in proliferation stage. And then cell immunohistochemistry identified Oct-4 positive cells, neurons and astrocytes, flow cytometry tested the Oct-4 positive cell percentage, and the apoptosis testing kit tested the cell apoptosis. Results： The Oct-4 positive cell percentage of mouse ES derived progenitors depressed to 4.33 （1.63% from 16.17（4.8%, cell apoptosis rate of serially low and high density passage in differentiated expanding stage are respective 15. 16 （ 3. 64% and 11. 88 （ 2. 63%, and cell immunohistochemistry identified that GFAP and Tuj-1 of the differentiated cells are negative. Conclusion： The Oct-4 positive cell percentage of mouse ES derived progenitors decreased significantly in this differentiation system, and it＇ s not due to other cells apoptosis was testified. Unexpectedly, the differentiation ability of the progenitors into GFAP positive astrocytes and Tuj-1 positive neurons decreased dramatically at the same time. This result suggested that high density passage play an important part in process of the progenitors differentiated into neurocytes, and the comparision between high and low density culture supplied new platform and the development direction of the neurons differentiation mechanism from ES.