中文摘要：

目的：构建高表达糖原合成激酶3β（GSK3β）的SH—SY5Y（人骨髓神经母细胞瘤细胞）转基因细胞模型，观察GSK3β高表达对宿主细胞tau蛋白磷酸化、微管稳定性的影响。方法：构建GSK3β真核表达质粒，转染SH—SY5Y细胞，使GSK3β在SH—SY5Y细胞中得到高表达，应用蛋白免疫印迹方法，观察tau蛋白磷酸化、总tau蛋白、微管蛋白乙酰化水平的变化情况。结果：GSK3β真核表达质粒转染SH—SY5Y细胞36h后，GSK3β在SH—SY5Y细胞中的表达达到高峰，tau蛋白Ser^199/202、Thr^231、Thr^205等位点的磷酸化水平在转染后48h达到高峰；tau蛋白总量未有明显变化。转染后60h，微管蛋白乙酰化水平明显减低。结论：高表达GSK313的SH—SY5Y细胞可使tau蛋白的磷酸化水平增高，从而降低tau蛋白结合和稳定微管蛋白的能力，导致微管蛋白乙酰化水平明显减低。

英文摘要：

AIM： To construct GSK3β-overexpressed SH -SYSY cells and to observe the effects of GSK3β-overexpression on tau protein phosphorylation and tubulin aeetylation in SH -SY5Y engineered cells. METHODS： The eDNA of GSK3β construct was subcloned into mammalian expression vector pBudCF4. 1. The integrity of the GSK3[3 con- struct was confirmed by sequence analysis. GSK3β was transiently transfected into SH -SYSY cells using Lipofectamine2000. Western blotting was used to measured protein levels of GSK3β and phosphorylating GSK3β, as well as, the total tau and phosphorylated tau protein and aeetylated tubulin. RESULTS： 36 h after transfection, the levels of GSK3β and phosphorylating GSK3β in SH -SY5Y cells were significantly increased compared with non -transfeetion group and vector group. After 48 h, the levels of phosphorylated tau protein （Ser^199/202, Thr^231 and Thr^205 residues） but not total tau protein were markedly increased in GSK313 -overexpressed SH -SY5Y cells. In addition, the level of acetylated tubulin was lower than that in non - transfection group and vector group. CONCLUSION： The over - expression of GSK3β in SH -SY5Y cells results in robust increases in tau protein phosphorylation at Ser^199/202, Thr^231 and Thr^205 residues, and de- creases in tubulin acetylation.