南方医科大学学报

南方医科大学学报 ›› 2016, Vol. 36 ›› Issue (07): 904-.

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ApoE4在U87细胞中可增加GSK-3β的表达及Tau的磷酸化

何颜结，卫佩如，伍巧燕，张馨宇，张兴梅，刘晓加，王方   

  • 出版日期:2016-07-20 发布日期:2016-07-20

ApoE4 increases glycogen synthase kinase 3β expression and Tau phosphorylation in U87 cells

  • Online:2016-07-20 Published:2016-07-20

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摘要： 目的探讨ApoE4 与GSK-3β及Tau 蛋白超磷酸化之间的关系，为研究ApoE4 在阿尔茨海默病（AD）中的作用机制提供 实验依据。方法分别将对照质粒载体pIRES-EGFP、重组质粒ApoE4/pIRES-EGFP 和ApoE3/pIRES-EGFP 转染U87 脑星形 胶质细胞系，用免疫蛋白印迹法（Western blotting）检测p-Tau/Tau 及GSK-3β蛋白水平的变化。在U87 细胞中转入干扰ApoE 表达的siRNA（ApoE-siRNA）及对照siRNA，检测p-Tau/Tau 及GSK-3β蛋白的改变情况。将质粒转染前后及siRNA干扰前后 目标蛋白的含量或磷酸化水平的改变进行统计学比较。结果与对照组相比，GSK-3β蛋白水平的相对值分别为：ApoE4 组 （1.819±0.130，P<0.01，n=3）和ApoE3 组（1.336±0.130，P<0.01，n=3），差异具显著差异，且ApoE4 和ApoE3 组比较也具有显 著性差异（P<0.01）。同样，相对于对照组，ApoE4 和ApoE3 组的磷酸化Tau 蛋白（p-Tau）的相对值分别为1.587±0.027（P< 0.01，n=3）和1.436±0.026（P<0.01，n=3），均具显著差异；且ApoE4 组较ApoE3 组的促进作用更强，两组间具显著差异（P< 0.01)。此外，用ApoE-siRNA 干扰后，U87 细胞中GSK-3β的表达和p-Tau 水平均较对照组显著降低，分别为0.544±0.058（P< 0.001，n=3）和0.474±0.060（P<0.01，n=3）。结论AD危险因子ApoE4 可能通过上调GSK-3β的表达而促进Tau 的磷酸化。

Abstract: Objective To explore the relations among apolipoprotein E4, Tau protein and glycogen synthase kinase 3β (GSK-3β). Methods U87 cells were transfected with pIRES-EGFP (control) or the recombinant plasmids ApoE4/pIRES-EGFP or ApoE3/ pIRES-EGFP, and the expression levels of p-Tau/Tau and GSK-3β in the cells were examined with Western blotting. To further confirm the effect of ApoE on GSK-3β and p-Tau expressions, a short interfering RNA (siRNA) targeting ApoE (ApoE-siRNA) was transfected into U87 cells via Lipofectamine 2000 and the protein expressions were examined 24 h later. Results Compared with those in the control group, the expressions levels of both GSK-3β and p-Tau/Tau increased significantly in the cells transfected with ApoE4 and ApoE3 plasmids (P<0.01), and the ApoE4 plasmid produced a more potent effect than the ApoE3 plasmid on the protein expressions (P<0.01). ApoE knockdown resulted in significantly reduced expressions of GSK-3β (P< 0.001) and p-Tau (P<0.01) in the cells. Conclusion ApoE4 can enhance Tau phosphorylation though upregulating GSK-3β, which sheds light on a new role of ApoE4 in Alzheimer’s disease.