Ser^(202)/Thr^(205)位点磷酸化tau在神经元中的定位  

Differential induction of tau phosphorylation by OA treatment in primarily cultured neurons and SH-SY5Y cells

作　　者：孙爱民[1,2] 王海萍[1] 沈玉君[2] 沈玉先[1,2] 方圣云[1] 

机构地区：[1]安徽医科大学基础医学院,安徽合肥230032 [2]安徽医科大学生物药物研究所,安徽合肥230032

出　　处：《中国药理学通报》2011年第1期41-45,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30772557;30870881)

摘　　要：目的观察Ser202/Thr205位点的过度磷酸化tau在神经元中的定位和聚集情况。方法取孕18 d SD胎鼠皮层神经元进行原代培养,于培养第9天加入冈田酸(okadaic acid,OA)处理12 h以诱导tau蛋白磷酸化;同时用OA诱导SH-SY5Y细胞;观察tau磷酸化后神经元形态的变化;用AT-8抗体免疫荧光染色观察Ser202/Thr205位点磷酸化tau的定位和聚集;同时应用AT-8抗体和Tau-5抗体检测tau蛋白Ser202/Thr205位点的磷酸化水平变化情况。结果经过Anti-NeuN和Anti-GFAP免疫染色鉴定,孕18 d胎鼠皮层神经元选择性原代培养成功。OA处理12 h以后,Ser202/Thr205位点磷酸化tau的水平明显增加,且出现高分子量的寡聚体;免疫荧光染色发现,Ser202/Thr205位点磷酸化的tau在原代培养神经元的胞质聚集;而OA处理的SH-SY5Y细胞,Ser202/Thr205位点磷酸化的tau却定位于细胞核。同时发现OA处理的原代神经元突起明显减少或消失。结论 OA能够诱导原代培养的胎鼠神经元tau蛋白在Ser202/Thr205位点过度磷酸化,且该磷酸化tau在不同类型或来源的神经元中的定位不同。Aim To investigate the characteristics of tau phosphorylation induced by OA treatment in primarily cultured embryonic neurons and SH-SY5Y cells.Methods Eighteen-day Sprague Dawley embryos were sacrificed to perform primary cultivation of cortical neurons.After treatment with OA for 12 h on day 9,the neurons were subject to immunocytochemistry and Western blot using Ser202/Thr205 phosphorylation-dependent antibody AT-8.Tau-5 antibody was also used as a counterpart to recongnize the total tau protein.Results The embryonic neurons were successfully cultivated and identified by double labeled immunofluorescent staining using anti-NeuN and anti-GFAP.Western blot analysis showed that the level of phosphorylated tau,recognized by AT-8,remarkably increased after OA treatment for 12 h.It was further confirmed by the bands detected by Tau-5 antibody,which recognized the total tau.Meanwhile,AT-8 detected a band with high molecular weight,suggesting a dimmer of phosphorylated tau.Immunostaining with AT-8 showed that OA induced tau phosphorylation and accumulation in the cytosol,compared with DMSO.However,OA-induced phosphorylated tau was also found localized in nuclei in SH-SY5Y.Consequently,the morphology of neurons was observed to be abnormal after OA treatment.Conclusion OA remarkably induces tau hyperphosphorylation at Ser202/Thr205 sites,which differentially accumulates in neurons.

关 键 词：冈田酸 胎鼠皮层神经元 tau 过度磷酸化 阿尔采末病 磷酸化位点 

分 类 号：R-332[医药卫生] R322.81