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目的探讨AKT-糖原合成激酶-3β(GSK-3β)信号通路对SOD1G93A突变N2a细胞的作用及机制。方法选取小鼠成神经瘤细胞系N2a,分别转染p EGFP-WT-SOD1和p EGFP-G93A-SOD1质粒,应用Western blotting和免疫荧光染色方法检测AKT、GSK-3β和细胞周期蛋白D1(cyclin D1)在细胞模型中的表达变化。应用RT-PCR和Western blotting技术检测siRNA沉默AKT后GSK-3β和cyclin D1在SOD1突变N2a细胞中的表达变化,通过MTS方法,检测细胞增殖和存活的改变。结果与p EGFP-WT-SOD1转染的N2a细胞比较,p EGFP-G93A-SOD1转染的N2a细胞中AKT及GSK-3β总蛋白在转染后24 h和48 h表达均无明显变化,p-AKT(Ser473)、p-GSK-3β(Ser9)和cyclin D1表达均升高。免疫荧光染色结果显示,转染后24 h和48 h,p-AKT(Ser473)、p-GSK-3β(Ser9)和cyclin D1在p EGFP-G93A-SOD1转染的N2a细胞中表达均升高。应用siRNA沉默AKT后与对照组相比,在转染后48 h和72h,AKT、p-AKT(Ser473)、GSK-3β、p-GSK-3β(Ser9)和cyclin D1蛋白均降低。MTS实验结果显示,在AKT沉默后72h、96 h、120 h,N2a细胞增殖和活力降低。结论 SOD1G93A突变影响N2a细胞中AKT、GSK-3β翻译后磷酸化修饰及cyclin D1的表达,AKT可能通过调控GSK-3β和cyclin D1影响SOD1G93A突变N2a细胞的增殖和存活。
Objective To investigate the effect and mechanism of AKT-glycogen synthesis kinase-3β (GSK-3β) signaling pathway on SOD1G93A mutant N2a cells. Methods The mouse neuroblastoma cell line N2a was transfected with pEGFP-WT-SOD1 and pEGFP-G93A-SOD1 plasmids respectively. Western blotting and immunofluorescence staining were used to detect the expressions of AKT, GSK-3β and cyclin D1 D1) Changes in expression in a cellular model. The expression of GSK-3β and cyclin D1 in SOD1 mutant N2a cells was detected by RT-PCR and Western blotting after AKT was silenced. The cell proliferation and survival were detected by MTS assay. Results Compared with pEGFP-WT-SOD1 transfected N2a cells, the expression of AKT and GSK-3βprotein in pEGFP-G93A-SOD1 transfected N2a cells had no significant change at 24 h and 48 h The expressions of -AKT (Ser473), p-GSK-3β (Ser9) and cyclin D1 increased. The results of immunofluorescence staining showed that the expressions of p-AKT (Ser473), p-GSK-3β (Ser9) and cyclin D1 were significantly increased in pEGFP-G93A-SOD1 transfected N2a cells at 24 h and 48 h after transfection . AKT, p-AKT (Ser473), GSK-3β, p-GSK-3β (Ser9) and cyclin D1 were all decreased at 48 h and 72 h after transfection compared with the control group. The results of MTS showed that the proliferation and the viability of N2a cells decreased at 72 h, 96 h and 120 h after AKT silencing. Conclusions SOD1G93A mutation affects posttranslational phosphorylation of AKT and GSK-3β and the expression of cyclin D1 in N2a cells. AKT may affect the proliferation and survival of N2a cells by regulating GSK-3β and cyclin D1.