神经干细胞体外分化抗原表达的研究

The Expression of Antigen in Neural Stem Cells Differentiation in vitro

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 explore the trends of nestin, SOX2, DCX, Tujl, GFAP and NeuN expressed in the process of neural stem cells differentiating into neurons in vitro in order to understand the factors affecting the neural stem cells differentiation process in vitro. Methods 12-14 day rat embryo was used as the source of neural stem cells for culture in vitro. Three days after the 1st passage, the neural stem cells were moved into specific culture medium for differentiation. Immunofluorescence methods were used to detect the nestin, SOX2, DCX, Tujl, GFAP and NeuN at different time points （dayl, day4, day7, dayl0, dayl4）. Results The expression of SOX2 and Nestin of the most cells at the beginning was similar, and then gradually decreased with the passage of time, but the expression of SOX2 lasted relatively longer than nestin. High expression of DCX appeared in dayl, day4 and dayT, and then gradually decreased; only a few DCX-positive cells in dayl4 were seen. A small number of Tujl positive cells in the day7 appeared, and then followed by increased expression. There was 38.27 % Tujl positive cells in day14. No NeuN positive cells advented in day1, day4 and day7, while NeuN positive cells ac- counted for 21.11% in dayl4. High expression of GFAP-positive cells appeared in dayl, and then gradually decreased. The decreasing of expression of GFAP-positive cells was slower than that of nestin, even if there were great number of GFAP-positive cells in dayl4. Conclusion The expression of specific antigens in the process of differentiation of neural stem cells in vitro is an interlaced process. The decrease in the expression of the undifferentiated cells was consistent with the increase in the expression of the differentiated cells.