Nac-1在神经干/祖细胞自我更新维持中的作用
Nac-1 is required for maintenance of multipotency in neural stem/progenitor cells

摘要：目的 研究Nac-1在神经干/祖细胞(NSPCs)自我更新维持中的作用及其机制。方法 以胚胎干细胞(ESCs)来源的NSPCs为细胞模型，利用RNA干扰法敲低Nac-1表达并通过定量RT-PCR和Western blot检测干扰效率；通过细胞计数和流式细胞技术，检测NSPCs的增殖和凋亡情况；并利用荧光素酶等实验检测Nac-1对c-Myc的转录调控作用。结果 Nac-1在NSPCs中高表达，分化后其mRNA水平下降了77%。与对照组相比，实验组(干扰组)NSPCs中Nac-1的mRNA水平显著下降，干扰效率分别是69%和66%。Nac-1敲低组的NSPCs增殖缓慢，凋亡增多，趋向于分化，c-Myc的mRNA水平降低46%和57%。荧光素酶分析显示，Nac-1敲低组中，c-Myc启动子的活性下降了24%和36%，提示Nac-1可以调节c-Myc基因的启动子活性。结论 Nac-1促进NSPCs增殖并抑制其分化，是NSPCs自我更新维持所必需；Nac-1促自我更新的作用可能通过调控c-Myc的转录而实现。
ABSTRACT: Objective To study the regulatory mechanism of Nac-1 on the self-renewal of neural stem/progenitor cells (NSPCs). Methods The expression level of Nac-1 was detected by using ESCs-derived NSPCs as the cell model. RNA interference was used to reduce the expression of Nac-1; the interference efficiency was detected by quantitative RT-PCR and Western blot. The proliferation and apoptosis of NSPCs were detected by cell counting and flow cytometry. Luciferase assay was used to detect the transcriptional regulation of Nac-1 on c-Myc. Results Nac-1 was highly expressed in NSPCs, and its mRNA level decreased by 77% after differentiation. Compared with that in the control group, the mRNA level of Nac-1 in the NSPCs of the experimental group was significantly decreased, and the interference efficiency was 69% and 66%, respectively. NSPCs with Nac-1 knockdown showed slow proliferation, increased apoptosis and tended to differentiate, and the mRNA level of c-Myc decreased by 46% and 57% in two Nac-1 knockdown groups, respectively. Luciferase assay showed that the transcriptional activity of c-Myc promoter decreased by 24% and 36%, respectively, suggesting that Nac-1 could regulate the promoter activity of c-Myc gene. Conclusion Nac-1 can promote the proliferation of NSPCs and inhibit their differentiation by regulating the transcription of c-Myc