摘要/Abstract

摘要： 目的 ·探讨在泛素蛋白酶体系统（ ubiquitin proteasome system，UPS）受损的帕金森病（ Parkinsons disease，PD）细胞模型中分子伴侣介导自噬（ chaperone-mediated autophagy，CMA）对 α-突触核蛋白低聚体水平的影响。方法 ·培养稳转野生型 α-突触核蛋白的人神经母细胞瘤 SK-N-SH细胞系，加入蛋白酶体抑制剂 lactacystin构建 PD细胞模型。通过 Western blotting检测 α-突触核蛋白低聚体、溶酶体相关膜蛋白 2A型（lysosome-associated membrane protein type 2A，LAMP2A）和相对分子质量为 70 000的热休克同源蛋白（ heat-shock cognate protein of 70 kDa，HSC70）水平；使用 LAMP2A siRNA抑制 CMA功能，检测对 α-突触核蛋白低聚体水平及细胞存活率的影响；通过免疫共沉淀观察 LAMP2A与 α-突触核蛋白低聚体的相互作用。结果 · PD细胞模型中 α-突触核蛋白低聚体表达水平升高，与 CMA功能密切相关的蛋白 LAMP2A和 HSC70水平应激性增加；抑制 LAMP2A表达， α-突触核蛋白低聚体水平进一步升高，细胞存活率降低，且通过免疫共沉淀检测到 LAMP2A与 α-突触核蛋白低聚体存在相互作用。结论 · PD细胞模型中， CMA是调节 α-突触核蛋白低聚体水平的途径之一，抑制其功能可进一步增加 α-突触核蛋白低聚体水平及细胞毒性作用。
关键词: 帕金森病, &, alpha, -突触核蛋白, 低聚体, 分子伴侣介导自噬
Abstract:
Objective · To investigate the effects of chaperone-mediated autophagy (CMA) on α-synuclein oligomers level in the Parkinsons disease (PD) cell model with impaired ubiquitin proteasome system (UPS). Methods · The PD cell model was establishedadding the proteasome inhibitor lactacystin in the SK-N-SH cell line stably transfected with wild type α-synuclein. The levels of α-synuclein oligomers, lysosome-associated membrane protein type 2A (LAMP2A) and 70 kDa heat shock homologous protein (HSC70) were detected using Western blotting. CMA function was inhibited with LAMP2A siRNA, and its effects on α-synuclein oligomers and cell viability were detected. Furthermore, the interaction of LAMP2A with α-synuclein oligomers was detectedimmunoprecipitation. Results · In the PD cell model, the levels of α-synuclein oligomers, and CMA related proteins, i.e. LAMP2A and HSC70, were increased. Inhibiting the of LAMP2A further increased α-synuclein oligomers level, while it decreased cell viability. Furthermore, LAMP2A could interact with α-synuclein oligomers. Conclusion · In the PD cell model, CMA is one of the pathways regulating α-synuclein oligomers level. Inhibiting CMA function can further increase the α-synuclein oligomers level and deteriorate cell survival.
Key words: Parkinsons disease (PD), &, alpha, -synuclein, oligomers, chaperone-mediated autophagy (CMA)


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