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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。
AIM: To investigate the mechanism of stress and chaperon (STCH) antagonism of beta-amyloid 25-35 and Aβ25-35 on human neuroblastoma cell line SH-SY5Y in vitro. Methods: SH-SY5Y cells were treated with Aβ25-35 (20μmol / L) at different time points, then the endoplasmic reticulum (ER) was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot Stress-related molecules and phosphorylation of c-Jun amino-terminal kinase (JNK) were detected by radioimmunoassay. The expression of STCH was detected by calcium imaging with acetoxymethyl ester of Fura-2AM (100 nmol / L Staurosporin causes calcium overload. Results: After treated with Aβ25-35, the expression of STCH mRNA was up-regulated in SH-SY5Y cells and began to rise at 3h and reached the peak at 12h, then decreased. Glucose regulated protein 94 (Grp94) was up-regulated at 6h, 9h reached its peak, then quickly dropped. Aβ25-35 treatment of SH-SY5Y cells can activate JNK pathway, resulting in increased phosphorylation, over-expression of STCH, can inhibit JNK phosphorylation. Overexpression of STCH significantly antagonized stauroposrine-induced increases in free calcium. CONCLUSIONS: STCH may protect neurons by antagonizing the increase of free calcium and inhibiting the JNK pathway, which will provide useful clues for finding neuroprotective strategies.