摘要
目的观察外源性tau蛋白的过度表达对非神经来源的293T细胞生长与分化的影响。方法构建带有GFP标签的表达人全长tau蛋白的质粒pEGFP-C1-tau,并转染293T细胞,通过荧光显微镜、激光共聚焦显微镜观察及免疫印迹等方法,观察tau的过度表达对293T细胞的生长与分化的影响。结果在瞬时转染的293T细胞中可以观察到,GFP全细胞分布,而GFP-tau主要定位于胞质。tau的过量表达可引起细胞形态改变,如突起减少或消失,细胞分裂受阻及多核巨细胞的形成等,最后导致细胞死亡。在稳定转染细胞中,tau蛋白的过度表达可诱导细胞出现凋亡的特征性改变。结论tau蛋白在非神经源性细胞中的过度表达可影响细胞的分裂、分化,并诱导细胞凋亡,提示tau蛋白有直接的细胞毒作用。
Aim To investigate the effects of overexpression of tall protein on non-neuronal cell growth and differentiation. Methods Tau cDNA was amplified by PCR and inserted into BamHI and EcoRI sites of pEG-FP-C1. pEGFP-CI-tau was transfected to 293T cells by using LipofectamineTM 2000. Fluorescent microscopy and confocal microscopy were used to observe cell morphology. Immunoblotting assay was used to identify tau protein using anti-tau antibody tau-5. Results After transfection with pEGFP-C1 vector and pEGFP-C1- tau, GFP expression was observed under fluorescent microscopy. However, the pattern of intracellular GFP distribution was different. GFP spreads through the whole cell in which the blank vector was transfected, while it is located mainly in cytosol in the cells that pEGFP-C1- tau harbored. The cells became round, axons shortened or even disappeared, and multinucleared giant cell formed 16- 24 h after transfection with tau. Overexpression of tau protein induced typically apoptotic characteristics, including chromatin condensation, and budding. Conclusions Overexpression of the tau prorein has a negative effect on cell tion, and it induces cell apoptosis, suggesting that overexpressed tau protein is toxic to cells.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2008年第3期365-369,共5页
Chinese Pharmacological Bulletin
基金
教育部新世纪优秀人才支持计划资助项目(NoNCET-04-0589)
教育部重点项目资助项目(No206067)
关键词
TAU蛋白
转染
基因表达
细胞形态
tau protein
transfection
gene expression
cell morphology
