miR-124抑制Notch通路促进神经干细胞增殖与分化

miR-124 facilitates the proliferation and differentiation of neural stem cells by inhibiting the Notch pathway

背景:如何在体外快速而高效的获得大量高纯度神经细胞是目前国内外细胞移植治疗领域所面临的一个巨大挑战和难题。目的:探讨miR-124是否通过调控Notch通路对神经干细胞增殖与分化产生影响。方法:分离胎鼠神经干细胞,利用免疫荧光法进行鉴定,采用RT-qP CR法检测神经干细胞分化1,2,4,7d后miR-124表达情况,利用瞬时转染法分别过表达和干扰miR-124后,采用MTT法和免疫印迹法检测ki-67蛋白表达水平观察细胞增殖情况,采用RT-qPCR法和免疫印迹法检测β-tubulinⅢ表达水平观察细胞分化情况,采用RT-qPCR法检测Notch通路相关蛋白HES1,HEY2和CCND1的mR NA表达。结果与结论:(1)神经干细胞在向神经细胞分化过程中,miR-124的表达水平显著升高;(2)过表达miR-124不仅能够促进细胞增殖与分化,同时对Notch通路相关蛋白起到了抑制作用;(3)抑制miR-124表达起到了与上述相反的作用;(4)以上结果提示miR-124可能通过抑制Notch通路促进神经干细胞的增殖与分化。

BACKGROUND:How to quickly and efficiently obtain enough pure neurons in vitro is a huge challenge and problem faced by the scientists in the cell-transplantation field.OBJECTIVE:To explore whether miR-124 can influence the proliferation and differentiation of neural stem cells by regulating the Notch pathway.METHODS:The fetal rat neural stem cells were isolated and identified by immunofluorescence.RT-qPCR was used to detect the expression of miR-124 after 1,2,4,and 7 days of neural stem cell differentiation.After overexpression and interference with miR-124 via transient transfection,the expression of ki-67 protein was detected by MTT assay and immunoblotting.RT-qPCR and immunoblotting were used to detectβ-tubulin III expression for determination of miR-124 effects on cell differentiation.RT-qPCR was used to detect the mRNA expression of Notch pathway-related proteins HES1,HEY2 and CCND1.RESULTS AND CONCLUSION:During the differentiation of neural stem cells into neurons,the expression of miR-124 was significantly upregulated.Moreover,miR-124 overexpression promoted the proliferation and differentiation of NSCs,and suppressed the expression of proteins in the Notch pathway.Silencing the miR-124 expression,however,achieved the opposite results.The above results suggest that miR-124 may promote the proliferation and differentiation of neural stem cells by inhibiting the Notch pathway.