细胞周期蛋白依赖性蛋白激酶-5过度表达对神经细丝磷酸化的影响

摘要

背景:阿尔茨海默病的主要神经病理学特征之一是由过度磷酸化的细胞骨架蛋白(如(o),神经细丝)组成的神经原纤维缠结,细胞周期蛋白依赖性蛋白激酶5可催化(o)蛋白的磷酸化,但神经细丝是否可被细胞周期蛋白依赖性蛋白激酶5磷酸化及其在阿尔茨海默病发病中的作用却知之甚少.目的:在细胞水平观察细胞周期蛋白依赖性蛋白激酶5过度表达对神经细丝磷酸化的影响.设计:随机对照实验.单位:华中科技大学同济医学院基础医学院生物化学与分子生物学系.材料:实验于2001-02/10在华中科技大学同济医学院基础医学院生物化学与分子生物学系实验室完成.观察对象为培养的鼠成神经瘤细胞株(N2a)细胞.方法:培养N2a细胞,分为2组,一组为转染组,一组为未转染组.转染组用脂质体转染技术将细胞周期蛋白依赖性蛋白激酶-5基因转入N2a细胞株中,并建立稳定表达细胞周期蛋白依赖性蛋白激酶-5的N2a/cdk-5细胞株,免疫沉淀法及酶活性测定检测细胞周期蛋白依赖性蛋白激酶-5活性,免疫荧光和免疫印迹技术检测它的表达和神经细丝的磷酸化状态.主要观察指标:两组细胞内神经细丝磷酸化的程度.结果:在N2a细胞株转染组中,细胞周期蛋白依赖性蛋白激酶-5表达增加,并使抗体SMI31显色增强,SMI32显色减弱,提示神经细丝被过度磷酸化.与此同时,细胞周期蛋白依赖性蛋白激酶-5酶活性较未转染组提高3.5倍.结论:提示细胞水平的细胞周期蛋白依赖性蛋白激酶-5过度表达会导致细胞周期蛋白依赖性蛋白激酶-5过度激活和神经细丝过度磷酸化,而过度磷酸化的神经细丝可能参与了阿尔茨海默病的病理过程.%BACKGROUND: One of the key neuropathological changes in Alzheimer disease is that neurofibrils over phosphorylated cytoskeletal protein (such as r and neurofilaments) composed of entwist together, and the phosphorylation of τ protein can be catalyzed by cyclin dependent kinase 5 (CDK5),however whether the phosphorylation of neurofilaments can be catalyzed by CDK5, as well as its role in the pathogenesis of Alzheimer diseases is less acknowledged.OBJECTIVE: To explore the role of over-expression of intracellular CDK5 in the phosphorylation of neurofilamentsDESIGN: Randomized controlled study.SETTING: Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: This study was conduced at Biochemical and Molecular Biological Department of Tongji Medical College, Huazhong University of Science and Technology between February and May 2001. In vitro cultured rat neuroblastoma cell strain (N2a) was adopted as subjects.METHODS: In vitro cultured N2a cells were divided into 2 groups, namely transfection group and non-transfection group. In transfection group,CDK5 gene was transfected into N2a cell line by using liposome transfection technique so as to obtain N2a/CDK5 cell line stably expressing CDK5, immune-precipitation and enzyme activity assay was used to detect the CDK5 activity, meanwhile immunofluorescence technique and immuneblot assay was used to detect CDK5 expression and phosphorylation of neurofilaments.MAIN OUTCOME MEASURES: Phosphorylation of neurofilaments in both groups.RESULTS: In transfection group of N2a cell line, CDK5 expression increased presented by deep coloration of SMI31 antibody and weak coloration of SMI32 antibody, implying hyper-phosphorylation of neurofilaments. Meanwhile, the activity of CDK5 was 3.5 times higher than that in non-transfection group.CONCLUSION: Intracellular over-expressison of CDK5 would lead to hyperactivity of CDK5 and hyper-phosphorylation of neurofilaments, however the hyper-phosphorylation of neurofilamentsmight invlove in the pathological development of AD.