小鼠胚胎大脑皮质活化和静息神经干细胞的初步分离与鉴定

Preliminary separation and identification of quiescent and activated neural stem cells in mouse embryonic cerebral cortex

目的:从小鼠胚胎大脑皮质中分离并鉴定静息和活化的神经干细胞。方法:通过流式细胞术,从胚胎14.5 d的小鼠大脑皮质中分选出大小不同的细胞团;利用免疫荧光技术检测神经干细胞标志物Pax6以及增殖标志物Ki67的表达变化;借助RT-qPCR分析两组细胞中干细胞标志基因Pax6、Oct4、Sox2和Nanog的表达情况;采用流式细胞术及RT-qPCR分析两组细胞所处的周期;体外培养对大、小细胞组进行增殖能力检测。结果:免疫荧光结果表明,分离出的两类细胞均表达Pax6,且大细胞组Ki67为阳性,小细胞组为阴性。细胞周期分析显示,小细胞组G0/G1期比例高于大细胞组,而G2/M期为0,且cyclin A和cyclin B的表达显著低于大细胞组(P<0.05)。体外培养显示,相对于大细胞,小细胞形成的微球数量少,速度慢;大、小细胞形成的微球消化后,Pax6和Ki67染色均阳性,且阳性率无显著差异(P>0.05)。结论:大、小细胞团分别为活化和静息的神经干细胞。活化的干细胞具有较强的增殖能力,而静息干细胞自我复制与增殖能力较差。在体外培养条件下,静息的神经干细胞可以被转激活。

AIM:To isolate and identify quiescent and activated neural stem cells from mouse embryonic cerebral cortex.METHODS:Two cell clusters derived from mouse cerebral cortex on embryonic day 14. 5 were separated by flow cytometry. The expression of stem cell marker Pax6 and proliferation marker Ki67 was examined by immunofluorescence. The mRNA expression of stem cell marker genes Pax6,Oct4,Sox2 and Nanog were detected by RT-qPCR. Cell cycle was analyzed by flow cytometry and RT-qPCR. Proliferation ability was investigated by in vitro cell culture.RESULTS:In both 2 groups,the cells expressed Pax6. Immunofluorescence staining of Ki67 in the big cell group was positive,while that in small cell group was negative. Cell cycle assay showed that the proportion of G0/G1 phase in the small cells was higher than that in the big cells,the G2/M phase proportion was 0,and the expression of cyclin A and cyclin B was lower than that in the big cells(P<0. 05). When cultured in vitro,the number of microspheres formed by the small cells was smaller and the formation speed was slower than those of the big cells. After digestion of microspheres,Pax6 and Ki67 staining of both big and small cells was positive,and the positive rates were not different(P>0. 05),indicating that the quiescent neural stem cells were activated.CONCLUSION:The 2 cell clusters are quiescent and activated state of neural stem cells. The activated stem cells have strong abilities of self-renewing and proliferation,while these abilities of quiescent stem cells are poor. The quiescent stem cells can translate into activated ones when cultured in vitro for a period.