马齿苋多糖对小鼠大脑神经细胞代谢损伤修复机制的研究

The Metobolism Regenerating Mechanism of Portulaca oleracea Polysaccharide on Mouse Brain Neurons

为探讨马齿苋多糖(Portulaca oleracea polysaccharide,POP)对小鼠大脑神经细胞代谢损伤的修复保护机制,采用原代培养小鼠大脑神经细胞的方法,分别用20μmol/L的β淀粉样蛋白(β-amyloid,Aβ)处理原代培养细胞、20μmol/L的Aβ加10 mmol/L的POP共同处理原代培养细胞;对处理的原代细胞进行倒置相差显微镜观察与PI-Hochest双染色成活分析,用Western blotting检测处理后的原代细胞P35和P25条带的表达;pEGFP-tau-s3和pcDNA-GSK-3β转染原代培养细胞,pEGFP-tau-s3和pcD-NA-GSK-3β加POP共同转染原代培养细胞,48 h后荧光观察和Western blotting检测GSK-3β的表达。结果显示,倒置相差显微镜下经20μmol/L Aβ处理的原代培养细胞出现了皱缩,部分细胞死亡;经PI-Hochest双染色细胞成活分析,Aβ和POP共同处理的原代培养细胞成活率明显高于只用Aβ处理的原代培养细胞;Western blotting检测到Aβ处理的细胞出现了P25条带,而Aβ加POP共同处理的细胞P25条带明显减弱;Western blotting检测发现,pEGFP-tau-s3和pcDNA-GSK-3β与POP共转染的细胞中,其tau蛋白的迁移率高于只转染pEGFP-tau-s3和pcDNA-GSK-3β的细胞tau迁移率。所以POP对小鼠大脑原代培养细胞Aβ损伤有修复保护作用,并能减少P35到P25的裂解和GSK-3β对tau的磷酸化。

The method of the primary culture of mouse brain neurons was applied to discuss the protective regenerating mechanism of Portulaca oleracea polysaccharide（POP）on the metabolism damage of mouse brain neurons..The cultured cells were treated with 20μmol/Lβ-amyloid（Aβ）,and with 20μmol/L Aβplus 10 mmol/L POP combined,respectively.The primarily-cultured cells were observed under an inverted phase contrast microscope.After being double-stained with PI-Hochest,the survival rate of these cells was analyzed.The P35 and P25 gene expression of the treated primarily-cultured cells were detected applying Western blotting. Then the primarily-cultured cells were transfected by pEGFP-tau-s3 and pcDNA-GSK-3β,and by pEGFP-tau-s3,pcDNA-GSK3βplus POP respectively.48 h after transfection,these cells were observed under fluorescence and the expression of GSK-3βwas detected applying Western blotting.The results showed that the primarily-cultured cells treated with 20μmol/L Aβbecame wizened and some of them died under the inverted phase contrast microscope.According to survival analysis of PI-Hochest double-stained cells,the survival rate of the cells co-treated with Aβand POP was significantly higher than that only treated with Aβ.The Aβ treated cells exhibited P25 bands by Western blotting detection,which apparently were weaker in co-treated cells.Through Western blotting,it was found that the tau protein＇s mobility was higher in the cells co-transfected with pEGFP-tau-s3,pcDNA-GSK-3β plus POP than that transfected with pEGFP-tau-s3 and pcDNA-GSK-3β.Therefore,POP had a protective function of regenerating Aβdamage in the primarily-cultured cells of mouse brain,and it could also decrease the cell dissociation from P35 to P25 cells and the tau protein＇s phosphorylation by GSK-3β.