高表达GPX_l对阿尔茨海默病细胞模型内P-CREB水平的影响

目的探讨细胞内高表达谷胱甘肽过氧化物酶(GPXl)能否提高阿尔茨海默病(AD)细胞模型中cAMP反应元件结合蛋白(CREB)的磷酸化水平。方法采用MTT法确定G418的筛选浓度,采用目的质粒与绿色荧光蛋白质粒共转染的方式将GPXl重组质粒和pLNCX空载体质粒转染至PC12细胞,以G418筛选浓度筛选出成功转染进GPXl重组质粒和pLNCX空载体质粒的细胞;采用MTT法确定β淀粉样蛋白(Aβ25-35)的最佳诱导浓度,建立AD细胞模型;以最佳Aβ25-35浓度分别诱导转染GPXl重组质粒组和转染pLNCX空载体质粒组细胞48h,采用免疫细胞化学方法检测诱导前后各组细胞内P-CREB的水平。结果在最佳Aβ25-35浓度诱导转染GPXl重组质粒组和转染pLNCX空载体质粒组前,两组细胞内的P-CREB水平无统计学差异(P>0.05);诱导48h后,两组细胞内P-CREB水平均显著降低(P<0.01),但转染GPXl重组质粒组细胞内的P-CREB水平仍明显高于转染pLNCX空载体质粒组的水平(P<0.01)。结论 Aβ25-35可导致细胞内P-CREB水平降低,转染GPXl重组质粒可在一定程度上逆转Aβ25-35所致的P-CREB水平降低。

The effects of the highly expressed glutathione peroxidase-1 on the phosphorylation of cAMP response element-binding protein in Alzheimer disease cell model

Objective To explore the effects of highly expressed glutathione peroxidase-1(GPXl) on the protein level of phosphorylated cyclic AMP response element-binding protein(CREB)in Alzheimer disease(AD) cell model.Methods The selected concentration of G418 was determined by MTT assay.GPXl recombinant plasmid and pLNCX empty vector plasmid were transfected into PC12 cells by using the target plasmid co-transfected with green fluorescent protein(GFP) plasmids.The cells,which were successfully transfected with GPXl recombinant plasmid and pLNCX empty vector plasmid,were screened out by the selected concentration of G418.The optimal inducing concentration of Aβ25-35 was determined by MTT assay and AD cell model was established.The GPXl/pLNCX/ PC12 group and pLNCX/ PC12 group were treated with optimal concentration of Aβ25-35 for 48 h,the level of P-CREB in cells was measured by immunocytochemistry method.Results Before the Aβ25-35 treatment,there was no significant difference in the P-CREB level between these two transfected cell groups(P>0.05).After Aβ25-35 treatment for 48 h,the level of P-CREB was decreased significantly in these two groups(both P<0.01),however,the level of P-CREB in the GPXl/pLNCX/ PC12 group was higher than that in the pLNCX/ PC12 group(P<0.01).Conclusions Aβ25-35 can down-regulate P-CREB expression in cells.The transfection of GPXl recombinant plasmid can up-regulate P-CREB expression to some extent.