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目的:观察体外神经干细胞向神经元分化过程中标志性抗原的表达,为进一步研究神经干细胞体外增殖分化的影响因素提供参照。方法:取12～14天胎鼠大脑皮层,体外培养神经干细胞;于第1次传代后第3天将培养的神经干细胞置于含有分化培养液的培养皿中进行分化;于不同分化时间点(1、4、7、10和14天)采用免疫细胞化学方法检测神经巢蛋白(nestin)、SOX2、doublecortin(DCX)、Tuj1、神经元核心抗原(NeuN)各抗原的表达。结果:nestin和SOX2在开始时表达量非常近似,几乎在所有的细胞中两者均同时表达,随时间的推移,两者表达量均逐渐下降,但SOX2表达时间相对nestin持续更长;DCX表达量在分化1周内较高且变化不大,随后开始下降,至分化14天时仅有少数细胞呈DCX阳性;Tuj1在分化7天时已有少量表达,随后逐渐增加,分化14天时有38.27%的细胞表达Tuj1;在贴壁分化的1周内未见NeuN阳性细胞,但至分化14天时,Ne-uN阳性细胞占21.11%;在贴壁分化第1天时即有大量GFAP阳性细胞,其表达量随分化时间延长而逐渐下降,但下降趋势较nestin缓和,至分化14天时仍有大量GFAP阳性细胞。结论:神经干细胞体外分化过程中抗原的表达是一个相互交错的过程,未分化状态标志性抗原表达的减少与分化状态标志性抗原表达的增加互相对应。
OBJECTIVE: To observe the expression of landmark antigens in the process of neural stem cells differentiating into neurons in vitro, so as to provide references for further study on the factors affecting the proliferation and differentiation of neural stem cells in vitro. Methods: The neural stem cells were cultured in vitro from 12 to 14 days in the cerebral cortex of the fetus. The neural stem cells were placed on the culture dish containing differentiation medium on the 3rd day after the 1st passage for differentiation. At different differentiation time points 1, 4, 7, 10, and 14 days). Immunocytochemistry was used to detect the expression of nestin, SOX2, doublecortin (DCX), Tuj1 and NeuN. Results: The expression levels of nestin and SOX2 were very similar at the beginning, and both of them were expressed in almost all cells. The expression of both of them decreased gradually with the passage of time, but the expression of SOX2 was longer than that of nestin. The expression of DCX The quantity increased slightly and little changed within 1 week of differentiation, then began to decline. Only a few cells showed DCX positive after 14 days of differentiation. Tuj1 was slightly expressed at 7 days of differentiation and then gradually increased. At 14 days of differentiation, 38.27% The cells expressed Tuj1; NeuN-positive cells were not seen within 1 week of adherent differentiation, but Ne-uN positive cells accounted for 21.11% after 14 days of differentiation; a large number of GFAP positive cells were observed on the first day of adherent differentiation, With the extension of differentiation time and gradually decreased, but the downward trend than nestin mitigation, to 14 days after differentiation, there are still a large number of GFAP positive cells. CONCLUSION: The expression of antigen in neural stem cells differentiated in vitro is a process of mutual stagnation. The decrease of the expression of the labeled antigen in undifferentiated state and the increase of the expression of labeled antigen in differentiation state correspond to each other.