XBP1調(diào)控膠質(zhì)瘤細胞氧化應激的課件

XBP1調(diào)控膠質(zhì)瘤細胞氧化應激的

實驗研究劉耀華趙世光陳曉豐梁元鄭秉杰哈爾濱醫(yī)科大學附屬第一醫(yī)院神經(jīng)外科ROSCancerCell14,December9,2008CatalaseSODExogenousROS:Thelaststrawbreaksthecamel'sback.TheAchilles’Heelofcancercells(CancerCell.2008;14(6):427-9

IF24.962)腫瘤，包括膠質(zhì)瘤可以產(chǎn)生高水平活性氧；腫瘤細胞的Akt過度激活，決定了其對活性氧敏感;大量活性氧可以殺傷腫瘤，很多化療藥物為活性氧誘導劑;調(diào)控膠質(zhì)瘤細胞氧化應激水平可能對其治療具有積極意義?；钚匝酰≧OS）與膠質(zhì)瘤研究背景HypoxiaROSNutrientsDeprivationOxidativeStressERStressProtectivemolecules?研究背景：內(nèi)質(zhì)網(wǎng)應激、氧化應激、XBP1XBP1XBP1CCAATantioxidantgenes(Catalase,SOD1,etc)transcriptsERSECCAATNF-YXBP1NF-YNF-YtranscriptstargetgenestranscriptstranscriptsXBP1?transcriptstranscripts實驗設計膠質(zhì)瘤細胞系U251MG進行RNA干擾，抑制XBP1基因表達后比較細胞對外源性活性氧H2O2的敏感性；流式細胞技術檢測ROS蓄積和線粒體膜電位；檢測比較細胞內(nèi)抗氧化分子表達水平；將細胞進行XBP1基因過表達，觀察能否起到相反的效果；應用啟動子突變分析探討XBP1促抗氧化分子轉(zhuǎn)錄活性的機制。實驗結(jié)果一對H2O2的敏感性比較*****H2O2處理后trypanblue檢測細胞生存情況*P<0.05,**P<0.01實驗結(jié)果一RandomSiXBP113.0100101102103104FL1-HH2O2-0.5100101102103104FL1-H60.3100101102103104FL1-H-C100101102103104FL1-H5.47.1100101102103104FL1-H-C100101102103104FL1-H29.4100101102103104FL1-HH2O2-0.5100101102103104FL1-H29.4流式細胞技術檢測線粒體膜電位（MMP）對H2O2的敏感性比較實驗結(jié)果二抑制XBP1特異性增強細胞對氧化應激的敏感性對活性氧誘導劑PTL的敏感性比較（*P<0.05,**P<0.01)*****實驗結(jié)果二抑制XBP1特異性增強細胞對氧化應激的敏感性對其它2種化療藥物誘導的細胞死亡沒有明顯區(qū)別VP16FK228實驗結(jié)果二XBP1缺失特異性增強細胞對氧化應激的敏感性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實驗結(jié)果三抑制XBP1細胞內(nèi)活性氧（ROS）明顯增高100101102FL1-HC3hC3h100101102FL1-HRandomDCFH-DA孵育細胞，流式細胞技術檢測細胞內(nèi)ROS堆積：*P<0.05,**P<0.0124.912.1(4.6)(3.8)SiXBP1****實驗結(jié)果三抑制XBP1細胞內(nèi)活性氧（ROS）明顯增高C

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4hP-P38P38C1h2h4h6hC1h2h4h6hRandomSiXBP1H2O2PTL實驗結(jié)果四XBP1與細胞抗氧化分子的表達SOD1CatalaseGPXTRX1GAPDHRandomXBP1抑制XBP1后細胞中抗氧化分子的mRNA水平(**P<0.01)**實驗結(jié)果四XBP1與細胞抗氧化分子的表達SOD1TRX1CatalaseGAPDHC

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9hCatalase14-3-3

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9hH2O2處理細胞后未見Catalase、SOD1和TRX1表達變化RandomSiXBP1實驗結(jié)果四XBP1與細胞抗氧化分子的表達C1h2h3hC1h2h3hATPP38P38H2O2Catalase特異性抑制劑AT預處理的影響0200400600FL2-AAT-H2O2.018M162%0200400600FL2-AATM10.90200400600FL2-ACM10.90200400600FL2-AH2O2M117.0%**P<0.01CatalaseU251XBP1過表達實驗結(jié)果五XBP1可增強Catalase轉(zhuǎn)錄活性XBP1b-actinSOD1**P<0.01H2O2mockXBP1實驗結(jié)果六XBP1可增強Catalase轉(zhuǎn)錄活性熒光素酶（luciferase）活性分析*P<0.05**P<0.01實驗結(jié)果六XBP1可增強Catalase轉(zhuǎn)錄活性Catalase啟動子突變**P<0.01**P<0.01**P<0.01實驗結(jié)果七XBP1促進NF-Y與Catalase啟動子結(jié)合凝膠電泳遷移分析（EMSA）實驗結(jié)果七XBP1促進NF-Y與Catalase啟動子結(jié)合染色質(zhì)免疫沉降小結(jié)CCAAT