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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 investigate whether the use of continuous low-density passage during differentiation and expansion can reduce the proportion of Oct-4-positive cells in precursor cells differentiated from mouse embryonic stem cells into neurons and their effect on neural differentiation. Methods: The mouse embryonic stem cells were differentiated into neurons by the “five-step method”, and then were passaged continuously for 10 generations by continuous low-density passage after the expansion period. Oct-4 positive cells, neurons and glial cells were identified by immunohistochemistry. The proportion of Oct-4 positive cells was detected by flow cytometry. Apoptosis was detected by apoptosis kit. Results: The flow cytometry showed that the percentage of Oct-4 positive cells decreased from 16.17 ± 4.8% to 4.33 ± 1.63% in the continuous low-density passage of proliferation phase, and the density of low-density passaged cells and normal high The density of passaged cells were 15.16 ± 3.64% and 11.88 ± 2.63%, respectively. The differentiation of GFAP and Tuj-1 in step 5 was negative. CONCLUSIONS: Low density subculture reduces the proportion of differentiated Oct-4 positive cells, and this decrease is not due to apoptosis in Oct-4 positive cells. At the same time, it may be because low-density subculturing induced the qualitative change of cells compared with high density or changed the microenvironment in which the precursor cells differentiated into neurons, leading to the inability of precursor cells to differentiate into nerve cells. These results suggest that high-density culture plays an important role in the differentiation of precursor cells into neurons. The comparison of high-density and low-density cultures provides a new platform and research direction for studying the mechanism of ES cell differentiation into neurons.