XBP1调控胶质瘤细胞氧化应激的.

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ROSCancerCell14,December9,2008CatalaseSODExogenousROS:Thelaststrawbreaksthecamel'sback.TheAchilles’Heelofcancercells(CancerCell.2008;14(6):427-9IF24.962)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HypoxiaROSNutrientsDeprivationOxidativeStressERStressProtectivemolecules?研究背景：内质网应激、氧化应激、XBP1XBP1XBP1CCAATantioxidantgenes(Catalase,SOD1,etc)transcriptsERSECCAATNF-YXBP1NF-YNF-YtranscriptstargetgenestranscriptstranscriptsXBP1?transcriptstranscripts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00.10.250.51020406080100RandomSiXBP1H2O2concentration(mM)survivalfraction(%oftotal)*****H2O2处理后trypanblue检测细胞生存情况*P0.05,**P0.01实验结果一RandomSiXBP113.0100101102103104FL1-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)05102040020406080100RandomSiXBP1PTLconcentration(uM)survivalfraction(%oftotal)*****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C01020304050randomSiXBP1celldeath(%oftotal)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-H100101102103104FL1-H6.7Random-PTL-20100101102103104FL1-H100101102103104FL1-H15.2SiXBP1-PTL-20100101102103104FL1-H100101102103104FL1-H41.5CRandom-FK228100101102103104FL1-H100101102103104FL1-H28.6Random-VP16100101102103104FL1-H100101102103104FL1-H26.9SiXBP1-FK228100101102103104FL1-H100101102103104FL1-H23.5SiXBP1-VP16100101102103104FL1-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(foldincrease)****实验结果三抑制XBP1细胞内活性氧（ROS）明显增高C1h2h3h4hRandomSiXBP1P-JNKJNK-1P-P38P38C1h2h3h4hP-P38P38C1h2h4h6hC1h2h4h6hRandomSiXBP1H2O2PTL实验结果四XBP1与细胞抗氧化分子的表达SOD1CatalaseGPXTRX1GAPDHRandomXBP1抑制XBP1后细胞中抗氧化分子的mRNA水平(**P0.01)randomSiXBP10.00.20.40.60.81.0catalasetranscripts(fold)**实验结果四XBP1与细胞抗氧化分子的表达SOD1TRX1CatalaseGAPDHC1h2h3hC1h2h3hRandomSiXBP1C3h6h9hCatalase14-3-3C3h6h9hH2O2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C1h2h3hC1h2h3hATPP38P38H2O2Catalase特异性抑制剂AT预处理的影响0200400600FL2-AAT-H2O2.018M162%0200400600FL2-AATM10.90200400600FL2-ACM10.90200400600FL2-AH2O2M117.0%CATH2O2AT+H2O2020406080100celldeath(%oftotal)**P0.01CatalaseU251XBP1过表达实验结果五XBP1可增强Catalase转录活性XBP1b-actinSOD1CmockXBP101234567ROS(foldincrease)**P0.01H2O2mockXBP1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)mockXBP1relativeactivity(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0246CCAATbox(2/3)CCAATbox2GCbox2wildtypemockXBP1relativeactivity(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CCAATCatalasegenetranscriptsCCAATNF-YNF-YXBP1transcriptstranscriptsCatalaseCatalaseCatalaseROSOxidativestressXBP1XBP1ROSROSOxidativestressOxidativestressCellDeath