内质网应激通过抑制PI3K/AKT/mTOR信号通路诱导多形性胶质母细胞瘤干细胞凋亡的机制  

Endoplasmic Reticulum Stress Induces Glioblastoma Multiforme Stem Cells Apoptosis Via Inhibiting PI3K/AKT/mTOR Pathway

作　　者：张保豫[1] 侯博[1] 何海勇[1] 李文胜[1] 梁朝峰[1] 叶卓鹏[1] 郭英[1] 

机构地区：[1]中山大学附属第三医院神经外科,广东广州510630

出　　处：《中山大学学报（医学科学版）》2016年第2期183-189,共7页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金(2014A030313189;2014A030310408);广东省科技计划项目(2014A020211013);中山大学青年教师培养项目(20120171110083)

摘　　要：【目的】探索内质网应激(ER stress)通过抑制PI3K/AKT/m TOR信号通路诱导多形性胶质母细胞瘤肿瘤干细胞(GSC)凋亡的机制。【方法】临床获取多形性胶质母细胞瘤肿瘤组织标本,剪碎、胰酶消化、过滤、去除红细胞后,培养GSC;免疫荧光技术检测GSC抗原CD133、GFAP、Nestin、A2B5的表达;caspase3/7细胞凋亡检测试剂盒检测衣霉素(Tun)和毒胡萝卜素(Thap)对GSC的凋亡作用及MTS法检测细胞增殖活性;同时Western blot分析凋亡蛋白caspase3,ER stress标志蛋白C/EBP同源性蛋白(CHOP)及PI3K/AKT/m TOR信号通路标志蛋白AKT、m TOR表达水平;使用si RNA沉默CHOP基因后,检测上述蛋白表达水平变化。【结果】1周后肿瘤干细胞球形成,并可以表达CD133、GFAP、Nestin、A2B5等GSC抗原。ER stress激活后,可诱导GSC凋亡及降低细胞增殖活性,CHOP表达上调而p-m TOR、p-AKT(Ser473)、p-AKT(Thr308)表达下调;相反,沉默CHOP基因后,ER stress被抑制,上述效应减弱。【结论】ER stress可能通过负性调节PI3K/AKT/m TOR信号通路诱导GSC凋亡。[Objective] To explore the mechanism of endoplasmic reticulum stress （ER stress） inducing glioblastoma stem cells （GSC） apoptosis via inhibiting PI3K/AKT/mTOR pathway. [Methods] Tumor samples were obtained from glioblastoma multiforme （GBM） patients after surgically resected specimens. Samples were washed, chopped, trypsinized, and filtered. The red cells were removed. The cells were cultured with tumour sphere medium （TSM） in vitro. GSC markers CD133, GFAP, Nestin and A2B5 were observed by immune-microscopy. After treating GSC （with/without transfecting with CHOP siRNA） by tunicamycin and thapsigargin, apoptosis induction was assessed by caspase 3/7 apoptosis assay; the percentage of cell viability was measured by MTS. The expression of caspase-3, C/EBP homologous protein （CHOP）, p-roTOR, p-AKT （Ser473） and p-AKT （Thr308） were examined by Western blot. [Results] The GSC spheres were formed in 7 days and the GSC markers CD133, GFAP, Nestin, and A2B5 were expressed. After activating ER stress, GSC cell death and apoptosis were induced significantly by up-regulatin caspase-3, CHOP and down-regulating p-mTOR, p-AKT （Ser473 and p-AKT （Thr308）. However, transfecting with CHOP siRNA, the effects were attenuated. [ Conclusion ] ER stress might induce GSC apoptosis via inhibiting PI3K/AKT/mTOR pathway.

关 键 词：多形性胶质母细胞瘤 肿瘤干细胞 内质网应激 凋亡 

分 类 号：R363[医药卫生—病理学]