蛋白磷酸酯酶抑制剂冈田酸对人神经母细胞瘤 SK-N-SH细胞系tau蛋白磷酸化水平的影响

观察蛋白磷酸酯酶-1和蛋白磷酸酯酶-2A的抑制剂冈田酸(okadaicacid,OA)对人神经母细胞瘤系SK-N-SH细胞tau蛋白磷酸化水平的变化,确定tau蛋白过度磷酸化细胞模型的合适剂量和时间。用不同剂量OA与SK-N-SH细胞共温育不同时间,用显微镜观察细胞形态变化,用Western印迹法检测磷酸化tau蛋白和非磷酸化tau蛋白在Ser202位点和Ser404位点磷酸化水平的变化。10～160nmol/LOA与SK-N-SH神经细胞温育3～24h,可引起细胞形态损伤呈剂量依赖性和时间依赖性的变化,起效剂量和时间为10nmol/L和3h。10nmol/LOA与SK-N-SH细胞温育6～24h,磷酸化tau蛋白Ser199/Ser202位点和Ser404位点的表达明显增高,非磷酸化tau蛋白Ser202位点和Ser404位点的表达明显降低,总tau蛋白含量无明显变化。OA可以作为很好的研究tau蛋白过度磷酸化的工具药,10nmol/LOA与SK-N-SH神经细胞共温育6h可以作为制备细胞模型的适宜条件。

Effects of Protein Phosphatase Inhibitor Okadaic Acid on Phosphorylation of Tau Protein in Human Neuroblastoma SK-N-SH Cells

To investigate the effects of protein phosphatase(PP-1 and PP-2A) inhibitor okadaic acid(OA) on human neuroblastom SK-N-SH cells,and to determine the best dosage and duration of OA treatment in this cellmodel.The diverse doses of OA were incubated with SK-N-SH cells for different periods of time.The changes incell morphology were observed by microscope.The phosphorylation and non-phosphorylation level of tau protein atSer202 site and Ser404 site were detected with Western blotting method.Incubation of 10-160 nmol/L OA with SK-N-SH cells for 3-24 h induced dose-and time-dependent changes in cell morphology,in which the starting dose andtime of OA effect were 10 nmol/L and 3 h respectively.When 10 nmol/L OA was incubated with SK-N-SH cells for6-24 h,the expression of phosphorylated tau protein at Ser199/Ser202 site and Ser404 site was increased,theexpression of non-phosphorylated tau at Ser202 site and Ser404 site was decreased,and the total tau protein did notshow obvious changes.OA is a good tool drug for studying hyperphosphorylation of tau protein,and 10 nmol/L OAincubation with human neuroblastoma SK-N-SH cells for 6 h can be taken as the proper conditions for developingthe cell model of tau hyperphosphorylation.