【作者】 曾育琦；

【导师】 陈晓春；

【作者基本信息】 福建医科大学 ， 神经病学， 2004， 硕士

【摘要】 目的:探讨β淀粉样蛋白的多肽片段Aβ25-35(beta-amyloid protein 25-35, Aβ25-35)对胎鼠皮层神经元Tau蛋白磷酸化水平的影响及其机制,建立阿尔茨海默病(Alzheimer’s disease, AD)样Tau蛋白过度磷酸化的细胞模型;研究人参皂甙Rb1和Rg1减轻Aβ25-35诱导的胎鼠皮层神经元Tau蛋白过度磷酸化的作用及其可能机制。 方法:选用孕期18±2d的清洁级SD(Sprague-Dawley, SD)大鼠,分离纯化胎鼠皮层神经元,通过MTT法观察不同浓度的凝聚态Aβ25-35作用不同时间对皮层神经元生长的影响。选择适宜浓度的凝聚态Aβ25-35,作用于皮层神经元不同时间,应用蛋白免疫印迹和免疫细胞化学染色的方法,观察Tau蛋白磷酸化、总Tau蛋白和糖原合成激酶3β(Glycogen synthase kinase 3β, GSK-3β)的表达情况,了解GSK-3β在Aβ诱导的皮层神经元Tau蛋白过度磷酸化中的作用。随后,用不同浓度的人参皂甙Rb1、Rg1或GSK-3β特异性抑制剂氯化锂(Lithium Chloride, LiCl)预处理皮层神经元24h后,再加入终浓度为20μmol/L凝聚态Aβ25-35,探讨人参皂甙Rb1和Rg1是否能抑制GSK-3β的活性而减轻Tau蛋白磷酸化水平。结果:20μmol/L以上浓度的凝聚态Aβ25-35可抑制皮层神经元的生长,该作用随着Aβ作用浓度的增加和作用时间的延长而增强。以20μmol/L的Aβ25-35作用于皮层神经元,Tau蛋白Ser396、Ser199/202、Thr231位点的磷酸化水平在3h或6h开始增高,12h达到高峰,24h后逐渐下降;总Tau蛋白的变化趋势与Tau<WP=6>蛋白磷酸化的改变大体一致。在Tau蛋白磷酸化达高峰时,GSK-3β的总量及其活性形式磷酸化GSK-3β的蛋白表达水平也增加。用不同浓度的人参皂甙Rb1、Rg1或GSK-3β特异性抑制剂LiCl预处理24h后,可减轻Aβ25-35所诱导的Tau蛋白过度磷酸化,同时GSK-3β的总量及其活性形式磷酸化GSK-3β的蛋白表达水平也下降,且以10μmol/L Rb1和20μmol/L Rg1预处理后,对降低Tau蛋白磷酸化水平和GSK-3β蛋白表达水平的作用最为明显。 结论:20μmol/L凝聚态Aβ25-35可使胎鼠皮层神经元Tau蛋白在Ser396、Ser199/202、Thr231位点的磷酸化水平增高,成功地建立AD样Tau蛋白过度磷酸化的细胞模型;而GSK-3β是Aβ25-35诱导Tau蛋白过度磷酸化的关键分子,Aβ25-35主要通过激活GSK-3β从而使Tau蛋白的磷酸化水平增高。一定剂量的人参皂甙Rb1和Rg1可抑制GSK-3β的活性,最终达到减轻凝聚态Aβ25-35所诱导的胎鼠皮层神经元Tau蛋白过度磷酸化的作用。更多还原

【Abstract】 Objective To investigate the effect and the molecular mechanism of aggregated beta-amyloid protein25-35 (Aβ25-35) on the level of tau protein phosphorylation in embryo rat cortical neurons, and to explore the protective effects of gisenoside Rb1 and gisenoside Rg1 on Aβ25-35-induced tau protein hyperphosporylation. Methods Primary cultures of cortical neurons were prepared from the embryonic day 18±2 Sprague-Dawley rats. MTT assay was used to measure the cytotoxicity of Aβ25-35 in cortical neurons, then chose an appropriate concentration of Aβ25-35. In order to detect the levels of tau phosphorylation,total tau protein and glycogen synthase kinase-3β(GSK-3β), western blot and immunocytochemical staining were performed on neurons exposed to 20μmol/L Aβ25-35 in the presence or absence of 24-hour pretreatment with several dose of ginsenoside Rb1, ginsenoside Rg1 or 10mmol/L lithium chloride (LiCl), a specific inhibitor of GSK-3β. Results Aβ25-35 treatment above the concentration of 20μmol/L resulted in decreasing of cell viability of cortical neurons in a dose-dependent manner. Aβ25-35 (20μmol/L) exposure of neurons for various periods induced tau protein hyperphorylation. The levels of tau protein phosphorylation in the sites of Ser396, Ser199/202, Thr231 and the amount of whole tau increased after 3h or 6h exposure and reached the maximum level after 12h exposure, then <WP=8>gradually declined after 24h exposure. Meanwhile, the expressions of the amount of whole GSK-3β and activated GSK-3β were also increased after Aβ25-35 (20μmol/L) exposure of neurons for 12h. Pretreatment with several dose of ginsenoside Rb1, ginsenoside Rg1 or 10mmol/L LiCl, a specific inhibitor of GSK-3β, markedly attenuated Aβ25-35-induced tau hyperphosphorylation and the expression of GSK-3β, and 10μmol/L Rb1 or 20μmol/L Rg1 showed the best protective effect on attenuating Aβ25-35-induced tau protein hyperphosphorylation.Conclusion GSK-3β activation by Aβ25-35 may lead to extensive tau phosphorylation. Gisenoside Rb1 and gisenoside Rg1 can attenuate Aβ25-35-induced tau protein hyperphosphorylation by inhibiting the activation of GSK-3β.更多还原