1、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ROSCancer Cell 14,December 9,2008CatalaseSODExogenous 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
2、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HypoxiaROSNutrients DeprivationOxidative StressER StressProtective molecules?研究背景：内质网应激、氧化应激、XBP1XBP1XBP1CCAATantioxidant genes(Catalase,SOD1,etc)transcriptsERSECCAATNF-YXBP1NF-YNF-Ytranscriptstarget genestranscriptstranscriptsXBP1?transcriptstranscripts实验设计 胶质瘤细胞
3、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00.10.250.51020406080100RandomSiXBP1H2O2 concentration(mM)survival fraction(%of total)*H2O2处理后trypan blue检测细胞生存情况 *P0.05,*P0.01实验结果一RandomSiXBP1
4、13.0100101102103104FL1-HH2O2-0.5100101102103104FL1-H60.3100101102103104FL1-H-C100101102103104FL1-H5.47.1100101102103104FL1-H-C100101102103104FL1-H29.4100101102103104FL1-HH2O2-0.5100101102103104FL1-H29.4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)0510204002
5、0406080100RandomSiXBP1 PTL concentration(uM)survival fraction(%of total)*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C01020304050randomSiXBP1cell death(%of total)VP16FK228实验结果二XBP1缺失特异性增强细胞对氧化应激的敏感性9.0100101102103104FL1-H100101102103104FL1-HRandom-C.001SiXBP1-C100101102103104FL1-H10010110210310
6、4FL1-H6.7Random-PTL-20100101102103104FL1-H100101102103104FL1-H15.2SiXBP1-PTL-20100101102103104FL1-H100101102103104FL1-H41.5CRandom-FK228100101102103104FL1-H100101102103104FL1-H28.6Random-VP16100101102103104FL1-H100101102103104FL1-H26.9SiXBP1-FK228100101102103104FL1-H100101102103104FL1-H23.5SiXBP1-VP
7、16100101102103104FL1-H100101102103104FL1-H21.3PTLFK228VP16实验结果三抑制XBP1细胞内活性氧（ROS）明显增高100101102FL1-HC3hC3h100101102FL1-HRandomDCFH-DA孵育细胞，流式细胞技术检测细胞内ROS堆积：*P0.05,*P0.0124.912.1(4.6)(3.8)SiXBP101h2h3h0123456RandomSiXBP1ROS(fold increase)*实验结果三抑制XBP1细胞内活性氧（ROS）明显增高 C 1h 2h 3h 4hRandomSiXBP1P-JNKJNK-1P-P
8、38P38 C 1h 2h 3h 4hP-P38P38C 1h 2h 4h 6hC 1h 2h 4h 6hRandomSiXBP1H2O2PTL实验结果四XBP1与细胞抗氧化分子的表达SOD1CatalaseGPXTRX1GAPDHRandom XBP1抑制XBP1后细胞中抗氧化分子的mRNA水平(*P0.01)randomSiXBP10.00.20.40.60.81.0catalase transcripts(fold)*实验结果四XBP1与细胞抗氧化分子的表达SOD1TRX1CatalaseGAPDHC 1h 2h 3h C 1h 2h 3hRandomSiXBP1 C 3h 6h 9hC
9、atalase14-3-3 C 3h 6h 9hH2O2处理细胞后未见Catalase、SOD1和TRX1表达变化RandomSiXBP1实验结果四XBP1与细胞抗氧化分子的表达C1h 2h 3hC1h 2h 3hAT PP38P38H2O2Catalase特异性抑制剂AT预处理的影响0200400600FL2-AAT-H2O2.018M162%0200400600FL2-AATM10.90200400600FL2-ACM10.90200400600FL2-AH2O2M117.0%CAT H2O2AT+H2O2020406080100cell death(%of total)*P0.01Cat
10、alaseU251 XBP1过表达过表达实验结果五XBP1可增强Catalase转录活性XBP1b-actinSOD1CmockXBP101234567ROS(fold increase)*P0.01H2O2mock XBP1实验结果六XBP1可增强Catalase转录活性荧光素酶（luciferase）活性分析0204060vehicleCAT(-191/+68)CAT(-1394/+68)mockXBP1relative activity(fold)*P0.05*P0.01实验结果六XBP1可增强Catalase转录活性Catalase启动子突变0246CCAAT box(2/3)CCAA
11、T box2GC box2wild typemockXBP1relative activity(fold)*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CCAATCatalase genetranscriptsCCAATNF-YNF-YXBP1transcriptstranscriptsCatalaseCatalaseCatalaseROSOxidativestressXBP1XBP1ROSROSOxidativestressOxidativestressCellDeath
