中文摘要：

目的： p66Shc在线粒体内积累和HtrA2/Omi的功能缺陷都能导致线粒体损伤,诱导细胞凋亡。探讨在线粒体中HtrA2对p66Shc的调控作用。方法： 构建p66Shc和成熟型HtrA2的真核表达质粒,共转染HEK293T细胞,免疫印迹法（Western blot）检测p66Shc蛋白;构建原核表达质粒,大肠杆菌纯化蛋白,体外切割实验,SDS-PAGE分离后考马斯亮蓝染色检测;提取HtrA2功能缺陷小鼠（mnd2）大脑组织的线粒体,检测线粒体内p66Shc的蛋白水平。结果： 细胞实验和体外实验证明HtrA2可以切割p66Shc,且在mnd2小鼠大脑中,线粒体内p66Shc的蛋白水平明显升高（P〈0.05）。结论： p66Shc是HtrA2的直接底物,且HtrA2参与调节线粒体中p66Shc的蛋白水平,揭示了HtrA2发挥神经保护功能新的可能机制。

英文摘要：

Objective： Both accumulation of p66Shc in mitochondria and loss of Omi/HtrA2 lead to mitochondrial dysfunction, which triggers apoptosis. To study the regulation of HtrA2 to the mitochondrial pool of p66Shc. Methods： The p66Shc and mature HtrA2 cDNA were cloned into eukaryote expression vector, then the constructs were cotransfected into HEK293T cell line, analyze p66Shc by Western blot; Purify proteins by pET System, then conduct in vitro cleavage assay, SDS-PAGE and Coomassie brilliant blue staining; analyze mitochondrial p66Shc protein level in mammalian cells overexpressed full length HtrA2 or knocked down HtrA2; Analyze p66Shc protein level in mitochondria isolated from mnd2 mouse brain. Results： p66Shc was cleaved by HtrA2 in vivo and in vitro; in mammalian cells, overexpressing full length HtrA2 down regulate mitochondrial p66Shc, and knocking down HtrA2 up regulate mitochondrial p66Shc; In mnd2 mouse brain, the mitochondrial p66Shc protein level was higher than that of the wild type mouse（P〈0.05）. Conclusion： Omi/HtrA2 cleaves p66Shc, and regulates the protein level of mitochondrial p66Shc,suggesting a possible new mechanism of how Omi/HtrA2 protects neuronal cells.