【摘要】 目的在细胞水平研究cdk-5过度表达对神经细丝磷酸化的影响。方法 用脂质体转染技术将cdk-5基因转入N2a细胞株中,并建立稳定表达cdk-5的N2a/cdk-5细胞株,免疫沉淀法及酶活性测定检测cdk-5活性,免疫荧光和免疫印迹技术检测cdk-5的表达和神经细丝的磷酸化状态。结果 在N2a细胞株转染组中,cdk-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,cdk-5酶活性较未转染组提高3.5倍。结论提示细胞水平的cdk-5过度表达会导致cdk-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。更多还原

【Abstract】 Objective In this study, we investigated the effect of cyclin-dependent kinase (cdk-5) overexpression on neurofilament phosphorylation in cultured cells. Methods The cdk-5 was transfected into N2a cell line with cdk-5 by using Lipofectamine2000, the cdk-5 activity and neurofilament phosphorylation was detected by using immmunoprecipetation, cdk-5 activity assay, immun-ofluoresence and Western blots. Results It was found that overexpression of cdk-5 led to elevation of cdk-5 activity, which was 3. 5 times that of the control level. Overactivation of cdk-5 resulted in hyperphosphorylation of neurofilament. It was demonstrated that overexpression of cdk-5 caused reduced staining with antibody SMI32 and enhanced staining with antibody SMI31. Conclusion These results indicate that in vitro overexpression of cdk-5 could result in cdk-5 overactivation and neurofilament hyperphosphorylation, which might be the underlying mechanism of Alzheimer dis-更多还原