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目的建立稳定表达tau蛋白的稳转细胞系,并观察tau蛋白的过度磷酸化对稳转的非神经源性细胞形态和活性的影响。方法构建pEGFP-C1-tau真核细胞表达质粒,建立稳转和表达融合蛋白GFP-tau的293T细胞系,并用不同浓度的蛋白磷酸酯酶抑制剂冈田酸(OA)处理细胞以诱导其过度磷酸化,通过荧光显微镜观察细胞形态的变化,用MTT法监测细胞的活性及免疫印迹法检测tau蛋白的表达及磷酸化水平。结果成功地建立了稳定表达GFP-tau蛋白的非神经源性稳转细胞系,该细胞系在稳转后3~4周,细胞生长状态良好,细胞形态和突起生长基本正常。用OA处理后,细胞突起消失、变圆,贴壁性能差,死亡的细胞数量也增多,这些变化随着OA作用时间的延长或浓度的增加更加明显;MTT法显示细胞的增殖活性明显下降;免疫印迹法提示,100nmol/LOA作用8h后,磷酸化tau水平增加。结论对于稳定表达tau蛋白的非神经源性细胞,OA可降低其增殖活性,该作用可能与OA抑制细胞内去磷酸化过程有关。
Objective To establish a stable cell line stably expressing tau protein and observe the effect of tau hyperphosphorylation on stable non-neuronal cell morphology and activity. Methods The eukaryotic expression plasmid pEGFP-C1-tau was constructed and the 293T cell line stably transfected and expressing the fusion protein GFP-tau was established. Cells were treated with different concentrations of protein phosphatase inhibitor okadaic acid (OA) to induce their over-expression The changes of cell morphology were observed by fluorescence microscopy. The cell viability was monitored by MTT assay and tau protein expression and phosphorylation were detected by Western blotting. Results Non-neuronal metastatic cell lines stably expressing GFP-tau protein were established successfully. The cells grew well 3 to 4 weeks after the cells were stably transformed, and their cell morphology and process of cell growth were normal. After treatment with OA, cell protrusion disappeared, rounded, poor adherent performance, the number of dead cells also increased, these changes with the extension of OA time or concentration of the more obvious; MTT method showed a significant decrease in cell proliferation activity; Immunoblotting suggested that the level of phosphorylated tau increased after 100nmol / LOA treatment for 8h. Conclusion OA can reduce the proliferation activity of non-neuronal cells stably expressing tau, which may be related to the inhibition of intracellular OA by dephosphorylation.