摘要
目的:研究应激和分子伴侣蛋白(stress and chaperon,STCH)拮抗β淀粉样蛋白(beta-amyloid25-35,Aβ25-35)在体外对人神经母细胞瘤细胞系SH-SY5Y毒性的机制。方法:用Aβ25-35(20μmol/L)在不同时间点分别处理SH-SY5Y培养细胞,然后采用逆转录-聚合酶链式反应(RT-PCR)和蛋白印记(Western Blot)法检测内质网应激相关分子的表达和c-Jun amino-terminal kinase(JNK)的磷酸化,并采用负载钙荧光探针(acetoxymethyl ester ofFura-2,Fura-2AM)钙成像技术检测过表达STCH对100nmol/L星孢霉素(staurosporin)引起的钙超载的情况。结果:Aβ25-35处理SH-SY5Y细胞后引起STCHmRNA表达上调,在3h开始升高,12h达到高峰,然后下降;而葡萄糖调节蛋白94(glucose-regulated protein94,Grp94)则于处理后6h表达上调,9h达到高峰,然后快速下降。Aβ25-35处理SH-SY5Y细胞能够激活JNK通路,致其磷酸化增加,过表达STCH,能够抑制JNK的磷酸化。过表达的STCH能够显著拮抗stauroposrine引起的游离钙增加。结论:STCH可能通过拮抗游离钙增加,抑制JNK通路而发挥保护神经元的作用,这将为我们寻找神经保护的策略提供有益的线索。
Objective：To study the mechanism of stress and chaperon（STCH）attenuation of the neurotoxic effect of β-amyloid 25-35（Aβ25-35）on the human neuroblastoma SH-SY5Y in vitro.Methods：SH-SY5Y cultured cells were treated with Aβ25-35（20 μmol/L） at different time point.Then reverse transcription-polymerase chain reaction（RT-PCR） and Western Blot were performed to detect the endoplasmic reticulum stress-related molecule expression and c-Jun amino-terminal kinase（JNK） pathway’s phosphorylation.Further the calcium imaging with loading of acetoxymethyl ester of Fura-2（Fura-2AM） was carried out to examine the effect of STCH overexpression on the Ca2＋ overload triggered by 100 nmol/L of stauroporine.Results：Upregulation of STCH mRNA expression was shown after Aβ25-35 treatment,which began at 3 h,reached the peak at 12 h,then downregulated.While the mRNA expression of glucose regulated protein 94（Grp94） increased at 6 h,the highest at 9 h and then decreased.After Aβ25-35 treatment,JNK pathway was activated to induce the increase of JNK phosphorylation.Overexpression of STCH can attenuate it.Further overexpression of STCH can significantly decrease the free calcium level induced by staurosporine.Conclusion：STCH may inhibit JNK pathway and buffer calcium overload to protect neurons.These data may provide useful clue to searching new neuroprotective strategy.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2010年第6期643-646,共4页
Chinese Journal of Neuroanatomy
基金
上海市科委自然科学基金(07ZR14122)
