【作者】 陈辉；

【导师】 王廷华； 陆地；

【作者基本信息】 昆明医学院 ， 人体解剖学与组织胚胎学， 2006， 硕士

【摘要】 【目的】为探讨ROS在脊髓损伤病理发生过程中的作用及其分子机制，一定浓度H₂O₂作用于PC12细胞模拟其氧化应激状态，制作细胞凋亡模型；通过P13K特异性抑制剂LY294002预处理，研究PI3K／Akt信号通路活化在H₂O₂诱导的PC12细胞凋亡中的作用及其作用机制，为脊髓损伤临床治疗（如抗氧化治疗、筛选ROS相关信号通路新药物靶点）提供新途径。 【方法】不同浓度H₂O₂作用于PC12细胞24h，及H₂O₂作用前用PI3K特异性抑制剂LY294002（20μM）预处理1h，通过MTT法检测，LY294002预处理前后不同浓度H₂O₂处理对细胞增殖抑制率的影响；分别用100μM和200μMH₂O₂作用于PC12细胞24h，制作细胞凋亡模型；经Hoechst33342／PI核荧光双染、DNA片段化分析和流式细胞术检测LY294002预处理对细胞凋亡的影响，以确定P13K活化在H₂O₂诱导的PC12细胞凋亡的作用；为进一步研究PI3K／Akt信号通路的作用机制，利用Western blot技术检测LY294002预处理前后凋亡相关蛋白CPP32、凋亡调节蛋白Bcl-2家族中Bcl-2、Bad、和Bcl-X_L蛋白表达变化及Bad磷酸化情况。 【结果】1．MTT结果显示，50～200μM H₂O₂处理PC12细胞增殖抑制率随处理浓度增加而递增；LY294002预处理后细胞增殖抑制率明显高于相应的H₂O₂处理组。2．用H₂O₂（100μM或200μM）处理细胞24h，用Hoechst33342／PI核双染，荧光显微镜下观察，部分细胞核呈现典型的凋亡特征；LY294002预处理使凋亡细胞数明显增加，从（17.5±1.3）％和（29±2.1）％增加到（31±2.6）％和（44±3）％（P＜0.05）；DNA凝胶电泳显示特征性“梯状条带”，且LY294002更多还原

【Abstract】 Objective To investigate the effects and mechanism of phosphatidylinositol3-kinase（PI3K） signaling pathway on apoptosis induced by hydrogen proxide （H₂O₂） in rat pheochromocytoma（PC12） cells.Methods In the present study, PC12 cells were preconditioned with 20μMLY294002（a PI3K-specific inhibitor） for 60 min, followed by exposures to different concentrations （20, 50, 100, 200, 400, 500μM） of H₂O₂ for 24-h respectively, cells growth inhibition rate was assessed by MTT. Using H₂O₂（200μM）-induced apoptosis model in PC12 cells, pretreatment with or without LY294002（20μM） for lh, apoptotic cell death was assessed by Hoechst33342/PI nuclei double staining, DNA ladder and flow cytometry. To further explore the downstream effector of PI3K activation, activation of CPP32, protein expression related with apoptotic regulation including Bcl-2, Bcl-X_L, Bad, and phosphorylation of Bad was examined by Western blotting.Results After pretreated with LY294002（20μM） for 1h, ① by MTT, cellgrowth inhibition rate H₂O₂-caused was increased markedly was examined; ② typically apoptotic nuclei and more stronger DNA ladder was observed, increased in the number of apoptotic cell death was examined. （3） elevated level of CPP32 activation was observed, with decrease in protein expression of Bcl-2、 Bcl-X_L, and declined level in phosphorylation of Bad.Conclusions The results suggested, transient activation of PI3K prevents from apoptosis H₂O₂-induced in PC12 cells. PI3K signaling pathway delays the更多还原