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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,XBP1,调控胶质瘤细胞氧化应激的实验研究,刘耀华 赵世光 陈晓丰 梁元 郑秉杰,哈尔滨医科大学附属第一医院 神经外科,ROS,Cancer Cell 14, December 9, 2008,Catalase,SOD,Exogenous ROS: The last straw breaks the camels back.,The Achilles Heel of cancer cells(Cancer Cell. 2008;14(6):427-9,IF 24.962,),肿瘤，包括胶质瘤可以产生高水平活性氧；,肿瘤细胞的,Akt,过度激活，决定了其对活性氧敏感,;,大量活性氧可以杀伤肿瘤，很多化疗药物为活性氧诱导剂,;,调控胶质瘤细胞氧化应激水平可能对其治疗具有积极意义。,活性氧（,ROS,）与胶质瘤,研究背景,Hypoxia,ROS,Nutrients Deprivation,Oxidative,Stress,ER,Stress,Protective molecules,?,研究背景：内质网应激、氧化应激、,XBP1,XBP1,XBP1,CCAAT,antioxidant genes,(Catalase, SOD1, etc),transcripts,ERSE,CCAAT,NF-Y,XBP1,NF-Y,NF-Y,transcripts,target genes,transcripts,transcripts,XBP1,?,transcripts,transcripts,实验设计,胶质瘤细胞系,U251MG,进行,RNA,干扰，抑制,XBP1,基因表达后比较细胞对外源性活性氧,H2O2,的敏感性；,流式细胞技术检测,ROS,蓄积和线粒体膜电位；,检测比较细胞内抗氧化分子表达水平；,将细胞进行,XBP1,基因过表达，观察能否起到相反的效果；,应用启动子突变分析探讨,XBP1,促抗氧化分子转录活性的机制。,实验结果一,对,H2O2,的敏感性比较,*,*,*,H2O2,处理后,trypan blue,检测细胞生存情况,*,P0.05, *P0.01,实验结果一,Random,SiXBP1,13.0,10,0,10,1,10,2,10,3,10,4,FL1-H,H2O2-0.5,10,0,10,1,10,2,10,3,10,4,FL1-H,60.3,10,0,10,1,10,2,10,3,10,4,FL1-H,-C,10,0,10,1,10,2,10,3,10,4,FL1-H,5.4,7.1,10,0,10,1,10,2,10,3,10,4,FL1-H,-C,10,0,10,1,10,2,10,3,10,4,FL1-H,29.4,10,0,10,1,10,2,10,3,10,4,FL1-H,H2O2-0.5,10,0,10,1,10,2,10,3,10,4,FL1-H,29.4,流式细胞技术检测线粒体膜电位（,MMP,）,对,H2O2,的敏感性比较,实验结果二,抑制,XBP1,特异性增强细胞对氧化应激的敏感性,对活性氧诱导剂,PTL,的敏感性比较（*,P0.05, *P0.01),*,*,*,实验结果二,抑制,XBP1,特异性增强细胞对氧化应激的敏感性,对其它,2,种化疗药物诱导的细胞死亡没有明显区别,VP16,FK228,实验结果二,XBP1,缺失特异性增强细胞对氧化应激的敏感性,9.0,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,Random-C.001,SiXBP1-C,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,6.7,Random-PTL-20,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,15.2,SiXBP1-PTL-20,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,41.5,C,Random-FK228,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,28.6,Random-VP16,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,26.9,SiXBP1-FK228,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,23.5,SiXBP1-VP16,10,0,10,1,10,2,10,3,10,4,FL1-H,10,0,10,1,10,2,10,3,10,4,FL1-H,21.3,PTL,FK228,VP16,实验结果三,抑制,XBP1,细胞内活性氧（,ROS,）明显增高,10,0,10,1,10,2,FL1-H,C,3h,C,3h,10,0,10,1,10,2,FL1-H,Random,DCFH-DA,孵育细胞，,流式细胞技术检测细胞内,ROS,堆积：,*,P0.05, *P0.01,24.9,12.1,(4.6),(3.8),SiXBP1,*,*,*,实验结果三,抑制,XBP1,细胞内活性氧（,ROS,）明显增高,C,1h,2h,3h,4h,Random,SiXBP1,P,-,JNK,JNK-1,P,-,P38,P38,C,1h,2h,3h,4h,P,-,P38,P38,C 1h 2h 4h 6h,C 1h 2h 4h 6h,Random,SiXBP1,H2O2,PTL,实验结果四,XBP1,与细胞抗氧化分子的表达,SOD1,Catalase,GPX,TRX,1,GAPDH,Random XBP1,抑制,XBP1,后细胞中抗氧化分子的,mRNA,水平,(*P0.01),*,实验结果四,XBP1,与细胞抗氧化分子的表达,SOD1,TRX1,Catalase,GAPDH,C,1,h,2,h,3,h,C,1,h,2,h,3,h,Random,SiXBP1,C,3h,6h,9h,Catalase,14-3-3,C,3h,6h,9h,H2O2,处理细胞后未见,Catalase,、,SOD1,和,TRX1,表达变化,Random,SiXBP1,实验结果四,XBP1,与细胞抗氧化分子的表达,C,1,h 2h 3h,C,1,h 2h 3h,AT,PP38,P38,H2O2,Catalase,特异性抑制剂,AT,预处理的影响,0,200,400,600,FL2-A,AT-H2O2.018,M1,62%,0,200,400,600,FL2-A,AT,M1,0.9,0,200,400,600,FL2-A,C,M1,0.9,0,200,400,600,FL2-A,H2O2,M1,17.0%,*P0.01,Catalase,U251 XBP1,过表达,实验结果五,XBP1,可增强,Catalase,转录活性,XBP1,b,-actin,SOD1,*,P0.01,H2O2,mock,XBP1,实验结果六,XBP1,可增强,Catalase,转录活性,荧光素酶（,luciferase,）活性分析,*P0.05,*P0.01,实验结果六,XBP1,可增强,Catalase,转录活性,Catalase,启动子突变,*P0.01,*P0.01,*P0.01,实验结果七,XBP1,促进,NF-Y,与,Catalase,启动子结合,凝胶电泳迁移分析（,EMSA,）,实验结果七,XBP1,促进,NF-Y,与,Catalase,启动子结合,染色质免疫沉降,小 结,CCAAT,Catalase gene,transcripts,CCAAT,NF-Y,NF-Y,XBP1,transcripts,transcripts,Catalase,Catalase,Catalase,ROS,Oxidative,stress,XBP1,XBP1,ROS,ROS,Oxidative,stress,Oxidative,stress,Cell,Death,
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