【摘要】 目的探讨溶酶体酶抑制剂对阿尔茨海默病(AD)相关蛋白Nicastrin(NCT)表达的影响,以期明确NCT的蛋白降解是否与溶酶体途径有关。方法在用人神经母细胞瘤细胞SH-SY5Y建立稳定表达NCT细胞株的基础上,应用溶酶体酶抑制剂处理NCT细胞株,并结合Western印迹、亚细胞器的分级分离、免疫荧光双标等技术,检测溶酶体酶抑制剂处理后神经细胞内NCT的表达变化。结果亚细胞器分级分离实验显示,正常情况下神经细胞内的NCT主要分布于内质网和高尔基复合体,少量NCT分布于溶酶体。Western印迹结果显示,溶酶体酶抑制剂处理后,神经细胞内源性和外源性成熟NCT(mNCT)的表达显著增强,且溶酶体酶抑制剂氯喹对mNCT蛋白表达的增强效应呈剂量依赖性和时间依赖性,但溶酶体抑制剂对非成熟NCT(imNCT)的表达无影响;免疫荧光双标结果显示,溶酶体酶抑制剂处理后,细胞内NCT的表达增强,且增多的NCT主要聚集在溶酶体内。结论神经细胞内mNCT的降解与溶酶体途径有关。更多还原

【Abstract】 Objective To explore whether the lysosomal pathway was involved in the degradation of Alzheimer’s disease(AD)-related protein Nicastrin(NCT).Methods Following generation of NCT stable cell lines,various methods such as Western blotting,double immunofluorescent staining and cell fractionation,combined with lysosomal inhibition were used to check NCT expression level in NCT stable cell line.Results Cell fractionation experiment showed that NCT distributed primarily in ER and Golgi apparatus,few NCT located in lysosome.Treatment of cells with lysosomal inhibitors significantly increased both endogenous and exogenous mature NCT(mNCT) in NCT stable cells or non-transfected neuronal cells,and the effect of lysosomal inhibitor on mNCT was time-and dose-dependent;however,lysosomal inhibitor had no effect on immature NCT(imNCT).Immunofluorescent microscopic analysis showed that lysosomal inhibition leaded to the accumulation of NCT in lysosomal apparatus.Conclusions The lysosomal pathway is involved in the degradation of mNCT in neuronal cells更多还原