Hippo-YAP信号通路在IFN-γ促神经干细胞增殖中的作用及其机制

The role and mechanism of Hippo-YAP signaling pathway in the IFN-γ-induced proliferation in NSCs

目的研究γ-干扰素(IFN-γ)对神经干细胞增殖及自我更新能力的影响;Hippo-YAP信号通路在IFN-γ促神经干细胞增殖以及自我更新能力的作用机制。方法从14~16 d胎鼠海马部提取分离神经干细胞进行体外培养,细胞免疫荧光Nestin染色确定神经干细胞分离成功。分为IFN-γ处理组和PBS对照组,计算神经球体数量及面积。CCK-8法测定神经干细胞增殖能力。实时荧光定量测定Hippo-YAP信号通路主要组分YAP的mRNA水平。蛋白免疫印迹法测定Hippo信号通路主要组分YAP蛋白、Cyclin D1蛋白水平。结果形态学研究显示IFN-γ处理组的神经干球数量及面积与PBS对照组相比增加,差异均有统计学意义(P<0.01);CCK-8试剂盒检测细胞活力显示IFN-γ处理好细胞活力比PBS组提高,差异有统计学意义(P<0.01)。实时荧光定量结果显示IFN-γ处理YAP的mRNA数量增加,差异有统计学意义(P<0.01);蛋白免疫印迹结果显示IFN-γ处理组的YAP蛋白、Cyclin D1蛋白增加,与PBS对照组相比差异有统计学意义(P<0.01)。结论 Hippo-YAP通路在IFN-γ促进神经干细胞的增殖及自我更新能力中发挥重要作用,主要机制是YAP蛋白激活Cyclin D1蛋白,促进神经干细胞的增殖及提高自我更新能力。

Objective To explore the effect of IFN-γ on the proliferation and self-renewal of neural stem cells and the mechanism of Hippo-Yap signaling pathway in the IFN-γ-induced proliferation in NSCs. Methods Neural stem cells harvested from the hippocampus tissue of 14~16 d mice were cultured. Immunofluorescence of Nestin was used to confirm NSCs were cultured. The IFN-γ and PBS solution were added to each group. The IFN-γ group and the PBS group were used to calculate the area and number of the neurospheres. Proliferation of neural stem cells was detected by CCK-8 kit. Real-time PCR was used to detect the expression of YAP mRNA. Western blot was used to detect the expression of YAP and Cyclin D1 proteins. Results The morphology revealed that the area and number of neurospheres were significantly increased by IFN-γtreatment(P<0.01);CCK8-kit showed that the cell viability of IFN-γ group was better than PBS group(P<0.01). Real-time PCR showed the mRNA expression of YAP in IFN-γ solution group was higher than PBS group;Western blot revealed the protein expression of YAP and Cyclin D1 in IFN-γ group were higher than those in PBS group,the difference was statistically significant(P<0.01). Conclusion IFN-γ enhanced the ability of self-renewal and proliferation of neural stem cells,and activated the Hippo-YAP signal pathway.