摘要
采用免疫组织化学、荧光双标记技术及大鼠额叶皮质定位注射OA的方法 ,观察和研究蛋白磷酸酶抑制剂冈田酸 (okadaicacid ,OA)对大鼠脑tau蛋白高度磷酸化和神经细胞退化的影响 ,结果表明 :①AT8免疫组织化学染色观察到在 2 0ngOA作用下神经细胞突起远端tau蛋白首先磷酸化 ,出现AT8即PHF tau免疫阳性反应 ,并逐渐向胞体发展 ,形成营养不良的神经细胞突起和神经纤维缠结样神经细胞 ;②细胞计数表明额叶皮质注射 2 0ngOA后 12hAT8免疫阳性神经细胞数显著增多 (P <0 .0 1) ,1d时达峰值 (P <0 .0 1) ,3d后减少 ;注射OA 2 0 ,5 0 ,10 0ng 1d时均大量表达AT8阳性细胞 (P <0 .0 5 ) ,但其各组间无显著差异 ;③免疫荧光双标记结果显示磷酸化tau蛋白在神经元和星形胶质细胞均存在 ,部分tau蛋白高度磷酸化的神经细胞TUNEL染色阳性 ,并伴有核浓缩断裂现象。这提示OA能有效诱导大鼠脑星形胶质细胞和神经元微管相关蛋白tau高度磷酸化 ,从而导致神经细胞DNA损伤 ;后者可能参与了OA诱导的神经细胞退化过程 ,其机制则有待阐明。
To study the effects of okadaic acid (OA), a protein phosphatase inhibitor, on tau protein hyperphosphorylation and neurodegeneration, OA in a 0.5 μl volume was injected into the frontal cortex of rat brain. The results showed that ① hyperphosphorylated tau protein caused by injection of 20 ng OA appeared in the distal axons and dentrites of neurocytes at 3 h following the injection, then distributed into cell bodies at 1 d to form the dystrophic neurites and neurofibrillary tangle like neurocytes by AT8, a tau protein phosphorylated marker, immuno reactive staining; ② the number of AT8 positive staining cells markedly increased at 12 h ( P <0.01), and peaked at 1 d ( P <0.01), then decreased at 3 d following the 20 ng OA injection; and that of different OA dosage at 1d also increased, but had no significant difference among 20, 50 and 100 ng OA groups;③ combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that AT8 positive staining appeared in neurons as well as astrocytes in the peri injected areas of the frontal cortex, and some of AT8 positive cells showed TUNEL immunopositive staining and nucleus pyknosis. These results reveal that OA can effectively induce tau protein hyperphosphorylation in the neurons as well as astrocytes in the peri injected areas of rat frontal cortex, and fewer neurocytes of tau protein hyperphosphorylation present the damage and fragmentation of the DNA double strands, suggesting that the damage of the DNA may play an important role in OA induced neurodegeneration, but the mechanism of the neurodegeneration remains to be further elucidated. \[
基金
国家自然科学基金 (3 982 5 10 9)
国家重点基础研究发展规划项目 (G19990 5 40 0 7)资助课题
