论文部分内容阅读
目的观察早老蛋白-1(Presenilin-1,PS1)的过度表达对tau蛋白磷酸化的影响。方法在NG-108细胞上转染不同的PS1质粒,利用免疫双标,免疫印记,免疫细胞化学,MTT,Western blot等方法,观察早老蛋白-1的过度表达对tau蛋白磷酸化的影响。结果免疫双标显示,在散发性AD(sporadic Alzheimer disease)患者脑内,PS1主要分布于神经元,并与磷酸化tau蛋白部分共存。免疫印迹显示,与空质粒组相比,转染野生型PS1组和转染一种家族性AD(familial Alzheimer disease,FAD)突变型早老蛋白-1(mPS1)质粒组的NG-108细胞中磷酸化的tau蛋白均增加。MTT方法未检测到这两组的转染细胞与对照组有显著差异。用Confocal显微镜观察PS1-EGFP质粒转染组,发现在转染后早期12 h,PS1-EGFP主要分布在细胞膜表面或细胞器上,随后PS1-EGFP主要在细胞浆中弥散分布。同时,免疫细胞化学检测显示,部分PS1-EGFP转染细胞中有明显的tau蛋白磷酸化,并且这些磷酸化tau蛋白能与PS1-EGFP蛋白一起形成聚集体。在给予磷酸酯酶抑制剂岗田酸后,PS1-EGFP转染细胞比未转染细胞含有更多的磷酸化的tau蛋白。结论野生型PS1可能参与了散发性阿茨海默氏病中的tau蛋白的病理改变。
Objective To observe the effect of Presenilin-1 (PS1) overexpression on tau phosphorylation. Methods The different PS1 plasmids were transfected into NG-108 cells. The effects of overexpression of presenilin-1 on phosphorylation of tau protein were observed by immunoblotting, immunocytochemistry, immunocytochemistry, MTT and Western blot. Results Immuno-double labeling showed that in the sporadic Alzheimer’s disease patients, PS1 mainly distributed in neurons and partially coexisted with phosphorylated tau protein. Western blotting showed that compared with the empty plasmid group, the transfection of wild-type PS1 group and NG-108 cells transfected with a familial Alzheimer’s disease (FAD) mutant type of presenilin-1 (mPS1) Tau protein are increased. MTT method did not detect the two groups of transfected cells and the control group were significantly different. PS1-EGFP plasmid transfected group was observed by Confocal microscope and found that PS1-EGFP mainly distributed on the cell membrane surface or organelles 12 h after transfection, PS1-EGFP was mainly distributed in the cytoplasm. Meanwhile, immunocytochemistry showed that some PS1-EGFP transfected cells had obvious tau phosphorylation, and these phosphorylated tau proteins could form aggregates together with PS1-EGFP protein. PS1-EGFP-transfected cells contained more phosphorylated tau protein than untransfected cells after administration of the phosphatase inhibitor glutamate. Conclusions Wild-type PS1 may be involved in the pathological changes of tau in sporadic Alzheimer’s disease.