去乙酰化酶Sirt1对神经母细胞瘤SK-N-SN细胞中Tau蛋白表达及磷酸化水平的影响

Effect of Sirt1 on the expression and phosphorylation of Tau protein in neuroblastoma SK-N-SH cells

目的观察Sirt1对神经母细胞瘤SK-N-SH细胞系中Tau蛋白磷酸化的影响。方法在体外培养的SK-N-SH细胞中加入腺病毒过表达的Sirt1和SirtM(Sirt mutant),利用Western blot检测细胞中Sirt1和SirtM的表达,并通过Western blot、Real-time PCR、免疫细胞化学技术检测t-Tau蛋白以及p-Tau蛋白的表达,流式细胞术(FCM)检测Sirt1对SK-N-SH细胞凋亡的影响。结果 Western blot结果显示,Sirt1组及SirtM组相比于对照组t-Tau蛋白的水平降低,Sirt1组较SirtM组相比降低较为显著;Tau蛋白的磷酸化位点ser404、thr231更为明显;Real-time PCR结果显示,Sirt1组相比于对照组Tau的mRNA水平下降;免疫细胞化学结果显示,Sirt1可以降低Tau蛋白及其磷酸化的水平;流式细胞术检测提示Sirt1组较对照组降低了SK-N-SH细胞的凋亡。结论Sirt1能够降低神经母细胞瘤SK-N-SH中Tau蛋白的表达及磷酸化、减少细胞的凋亡;本研究为Tau蛋白病等神经退行性疾病的研究提供了重要的实验依据。

Objective To observe the effect of Sirt1 on the phosphorylation of Tau protein in neuroblastoma SK-N-SH cell line.Methods We cultured SK-N-SH cells in vitro with adenovirus packaging of Sirt1 and SirtM(Sirt mutant),and then observed the expression of Sirt1 under an inverted fluorescence microscope.The expressions of Sirt1 and SirtM were detected by Western blot;t-Tau protein and phosphorylation of Tau protein were detected by Western blot,Real-time PCR,immunohistochemistry and immunofluorescence;and the effect of Sirt1 on SK-N-SH apoptosis was investigated by flow cytometry.Results The t-Tau protein level and its phosphorylation were significantly decreased in Sirt1 and SirtM groups compared with those in control group,and Sirt1 group showed more significantly decreased ser404,thr231 phosphorylation of tau protein and the mRNA level of Tau.Flow cytometry showed that Sirt1 could significantly reduce the apoptosis of SK-N-SH cells compared with the control group.Conclusion Sirt1 can decrease the phosphorylation of Tau protein and reduce the apoptosis of SK-N-SH,which provides an important laboratory basis for studies on Tau protein disease and other neurodegenerative diseases.