摘要
目的探讨磷酸酯酶活性降低致tau过度磷酸化与细胞凋亡的关系。方法稳定表达人tau cDNA的鼠成神经瘤细胞(N2a/tau)分3组,对照组不处理;十字孢碱(staurosporine,STP,凋亡诱导剂)处理6h为STP组;用磷酸酯酶PP-2A的抑制剂冈田酸(okadaic acid,OA)处理4h,再用STP处理6h为OA+STP组。流式细胞仪检测凋亡率,免疫印迹检测Ser396位点磷酸化tau(Ser396 p-tau)和总tau的表达,免疫荧光双标检测Ser396 p-tau的阳性产物与活化的半胱氨酸蛋白酶(Cleaved Caspase-3)阳性产物的共定位关系。结果①凋亡率:对照组为(4.37±1.47)%,STP组为(19.84±3.25)%,OA+STP组为(12.12±2.46)%;②免疫印迹:OA+STP组Ser396 p-tau的表达明显高于另两组,总tau表达组间无差异;③免疫荧光双标:进一步印证免疫印迹结果,且3组均显示活化的Caspase-3和Ser396 p-tau阳性产物大多不共定位于相同细胞。结论PP-2A活性降低所致tau Ser396位点的过度磷酸化可抑制细胞凋亡,结合前期的实验进一步明确特殊位点过度磷酸化的tau可抑制细胞进入快速的凋亡程序。
Objective To investigate the relationship between hyperpbosphorylation of tau caused by the reduced activity of protein phosphatase and agoptosis. Methods The mouse neuroblastoma N2a cells stably expressing human tau cDNA （N2a/ tau） were divided into three groups： the control group was not treated with special chemicals, the STP group was treated with staurosporine （STP） for 6 h, and the OA＋STP group with okadaic acid （OA） for 4 h, and then with STP for 6 h. The apoptosis rate was detected by using flow cytometry, the expression of phosphorylated tau with Ser396 （Ser396 p-tau） and total tau were examined by Western blot, and immunofluorescence double labeling technology was used to study the co-localization of cleaved Caspase-3 and Ser396 p-tau-positive product in cells. Results The apoptosis rate in control, STP, and OA＋STP groups was （4.37 ±1. 47）%, （19.84 ±3.25）%, and （12.12 ± 2.46）% respectively. Western blot results revealed that the expression level of Ser396 p-tau in OA＋STP group was significantly higher than in control and STP groups, but the total tau expression had no significant difference among three groups, Immunofluorescence results indicated that the cleaved Caspase-3 and Ser396 p-tau-positive product did not co-localize in the same cells in the three groups. Conclusion tau hyperphosphorylation at Ser396 induced by reduced activity of PP-2A inhibited apoptosis. This evidence and our previous results proved that hyperphos- phorylation of tau at special site inhibited the rapid process of apoptosis.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2009年第3期281-285,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金(No.30772292)
教育部博士点基金新教师基金(No.20070487104)资助项目
