CAJ | 학술논문

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.
목적 관찰조로단백-1(Presenilin-1,PS1)적과도표체대tau단백린산화적영향.방법 재NG-108세포상전염불동적PS1질립, 이용면역쌍표,면역인기, 면역세포화학,MTT,Western blot 등방법, 관찰조로단백-1적과도표체대tau단백린산화적영향.결과 면역쌍표현시,재산발성AD(sporadic Alzheimer disease)환자뇌내, PS1주요분포우신경원,병여린산화tau단백부분공존.면역인적현시, 여공질립조상비,전염야생형PS1조화전염일충가족성AD(familial Alzheimer disease,FAD)돌변형조로단백-1(mPS1)질립조적NG-108세포중린산화적tau단백균증가.MTT방법미검측도저량조적전염세포여대조조유현저차이.용Confocal현미경관찰PS1-EGFP질립전염조, 발현재전염후조기12 h,PS1-EGFP주요분포재세포막표면혹세포기상,수후PS1-EGFP주요재세포장중미산분포.동시,면역세포화학검측현시,부분PS1-EGFP 전염세포중유명현적tau단백린산화,병차저사린산화tau단백능여PS1-EGFP단백일기형성취집체.재급여린산지매억제제강전산후, PS1-EGFP 전염세포비미전염세포함유경다적린산화적tau단백.결론 야생형PS1가능삼여료산발성아자해묵씨병중적tau단백적병리개변.
Purpose To study the effect of overexpressing either wild type or a familial Alzheimer disease mutant presenilin 1 (mPS1) on tau phosphorylation in neuroblastoma NG-108 cells. Methods Three different plasmids transfected NG-108 cells respectively. Immunostaining and confocal microscopic technique were used to study the distribution of presenilin 1 and phosphorylated tau. Immunoblot test was applied to investigate the change of tau phosphorylation. Results Immunostaining showed that in brain of sporadic Alzheimer disease, PS1 mainly distributed in neuron and partially colocalized with the phosphorylated tau. Immunoblot tests showed that the cells transected either wild type PS1 or mPS1 contained more phorphorylated tau than the control cells. However, MTT test showed no significant difference between mock transfected cells and the wPS1 or mPS1 transfected cells. In addition, after transfection of the constructed PS1-EGFP vector, overexpressed EGFP-PS1 was located at cell surface membrane and subcellular organelles at earlier time at 12 hr, then EGFP-PS1 diffused in cytosol. Immunocytochemical observations demonstrated that some of the PS1-EGFP transfected cells contained more phosphorylated tau protein, which formed aggresome with PS-1-EGFP. When treated with phosphotase inhibitor okadaic acid, in the PS1-EGFP transfected cells accumulated more phosphorylated tau than the un-transfected cells. Conclusions Wild type PS1 is possibly involved in tauopathy in sporadic Alzheimer's disease.