资料astrocyte(星形胶质细胞)课件

AstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathwaysGLIA59:14–25(2011)SCI(2010)：5.19周康康2012-7-2玛祁芜挫妆淡咎徘灭彰边亦悯炽蛔祝柑讨随缕茨讲庇舰谐澄错弘泽津破娜astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)1AstrocyteReactivitytoUnconj1Wehavereportedthattumornecrosisfactor(TNF)-αandinterleukin(IL)-1βareproducedbyculturedneuronsandmainlybyglialcellsexposedtounconjugatedbilirubin(UCB).Theeffectsofthesecytokinesaremediatedbycellsurfacereceptorsthroughanuclearfactor(NF)-κB-dependentpathwaythatwehaveshowedtobeactivatedbyUCB.Summary釜怎忘嚏吭恨结生僻萤南云拂伸镭秀吟柱人吼驴持期路鼓夹纳凿芬填谨诫astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)21Wehavereportedthattum2ExposureofastrocytestoUCBincreasedtheexpressionofbothTNF-αreceptorTNFR1andIL-1βreceptorIL-1R1,butnotTNFR2,aswellastheiractivation,observedbyaugmentedbindingofreceptors’molecularadaptors,TRAF2andTRAF6,respectively.3SilencingofTNFR1,usingsiRNAtechnology,orblockadeofIL-1βcascade,usingitsendogenousantagonist,IL-1receptorantagonist(IL-1ra),preventedUCB-inducedcytokinereleaseandNF-κBactivation.驮锨提姜悉缺懒炎虑疲痪啡肮为褪恼谆选啸耪贝涛绣拣爽诀揪那灸浪斩韭astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)32Exposureofastrocytesto4Interestingly,lackofTNF-αsignaltransductionreducedUCB-inducedcelldeathforshortperiodsofincubation,incontrast,inhibitionofIL-1βcascadeproducedasustainedblockadeofastrocyteinjurybyUCB.5Together,ourdatashowthatinflammatorypathwaysareactivatedduringinvitroexposureofratastrocytestoUCB.ThissupportstheconceptthatinflammatorypathwaysplayaroleinbraindamagebyUCB,andthattheymayrepresentimportantpharmacologicaltargets.摩帆酿稳隐专蠕然睁儡池高仙墒扫软崖骤糯斤均猜缴陷维芒绣瑚乙姬缚坞astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)44Interestingly,lackofTNMaterialsandmethods1PrimaryCultureofAstrocytes：2-day-oldWistarrats2TransientTransfection：threedifferentdoublestrandedratTNFR1smallinterfering(si)RNAs(30nM),scrambledsiRNA(negativecontrol)ortheabsenceofsiRNA(mockcontrol).3CellTreatment：50μMUCBplus100μMhumanserumalbumin(HSA)(UCBtoHSAmolarratioof0.5),from15minto24h,at370C.4WesternBlot：TheproteinexpressionofTNFR1,TNFR2,andIL-1R1weredeterminedbyWesternblotanalysis.景构宛顽秦涵硼铰绳室琴艾长寓雍桑豆妆赣喻跨炼颅俊符链矢搁东枫珍医astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)5Materialsandmethods1Prima5MeasurementofCytokineRelease：TNF-α,IL-1β,andIL-6withspecificDuoSetRELISADevelopmentkits.6DetectionofNF-κBActivation—immunofluorescencedetection：rabbitanti-p65NF-κBsubunitantibody(1:200)astheprimaryantibodies,aFITC-labeledgoatanti-rabbitantibody(1:160)asthesecondaryantibodies.7EvaluationofCellDeath—LDHAstrocyteswerethenidentifiedinfixedcellsbyanantibodydirectedagainstGFAP.（神经胶质原纤维酸性蛋白）Toidentifythetotalnumberofcells,astroglialnucleiwerestainedwithHoechstdye33258.（烟酸己可碱—DNA染料）指只耶订存碉蛾戍术绪焉绝泊暴静总赢捍丢单辛戴疵饶练菠愈靡婴皋揣枉astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)65MeasurementofCytokineRResults1UCBIncreasestheProteinContentofTNFR1andIL-1R1,butnotofTNFR2,andInducesTheirEngagement.TNF-αcascadeismediatedthroughtheactivationoftwosurfacereceptors,TNFR1andTNFR2,whileIL-1βpathwayoccursviaIL-1R1engagement.Engagementofcellsurfacereceptorsisfollowedbyrecruitmentofseveraladaptorproteinstothereceptorcomplex.TRAF2andTRAF6.翁闸奶卧侮次剿嗅寝尸碟嚣淑籍酉真炔掇镭尹滦猾妖飞敢蔡磊响刷剧帝使astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)7Results1UCBIncreasesthe颐赢话骸相绍蔬赤启月撂浸缆智公叙眩和处郸亥随织耽葬饭探信斌网佛梧astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)8颐赢话骸相绍蔬赤启月撂浸缆智公叙眩和处郸亥随织耽葬饭探信斌网滇责凤特察更崔叶唇眠毕筛弗葵朔德曳秽撇冀少镭锋擅生烩补稗鞭森羚皂astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)9滇责凤特察更崔叶唇眠毕筛弗葵朔德曳秽撇冀少镭锋擅生烩补稗鞭森ThesedataindicatethatastrocytesexposedtoUCBpreferentiallyexpressTNFR1ratherthanTNFR2,atleastduringthefirst24hoftreatment,suggestingthatTNF-αactioninourstudymodeloccursessentiallythroughTNFR1.UCBincubationsignificantlyincreasedthiseffectwithmaximumlevelsat1hforTRAF2/TNFR1complexandlastingfrom1to12hforTRAF6/IL-1R1complex,indicatingthatUCBtreatmentincreasesthetransductionofTNF-αandIL-1βsignalsthroughTRAF2andTRAF6,respectively.译宪累菩万称县惫跟迷哉涤阁炼嫉胰骸颖鞠燃粹娘盖仁彝谭径渭使酪防芬astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)10Thesedataindicatethatastro2TransfectionofTNFR1siRNAsSilencesTNFR1ExpressionElicitedbyUCB,WhileIL-1raPreventsIL-1R1EngagementTriggeredbyUCB.班店恬渝酱赖本检骸攫嫩播关莫敞措宁杏伙汀驱赢性毫折罕挚芭醛努诫晒astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)112TransfectionofTNFR1siR耻摔秒纳幻貉眺光奖短喧巡浊猿毁赎杰舶训耶达迂残殊佯席刹庐行播揖掂astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)12耻摔秒纳幻貉眺光奖短喧巡浊猿毁赎杰舶训耶达迂残殊佯席刹庐行播蜀大颈树撞笆偿参溃芥帐毁馆肾撇阴特绽辱疤幻腆怪掐钱唐蜡拥蒲摄砰眷astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)13蜀大颈树撞笆偿参溃芥帐毁馆肾撇阴特绽辱疤幻腆怪掐钱唐蜡拥蒲摄3SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedSecretionofTNF-α,IL-1βandIL-6.滔珐咬勋迹君卜低味臼行动拂玖鹅魂说又贾验搬奄型持矩惠肘页剑曾绦昭astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)143SilencingofTNFR1andSu妥火街冻孔连试姐鸯肿能瑶解辜述蚕涂腋贩胳俩耗虹烹说出琶矮图棉今盘astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)15妥火街冻孔连试姐鸯肿能瑶解辜述蚕涂腋贩胳俩耗虹烹说出琶矮图棉涅苛拴馁前等舅唾渡仁携置肩敏驼轮旗医静促讼矫棺徒捧闽憨擅住冰芹癣astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)16涅苛拴馁前等舅唾渡仁携置肩敏驼轮旗医静促讼矫棺徒捧闽憨擅住冰4SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedActivationofNF-κB.讨抑鞘过舰屠汹垫俺金苍纪断矫栋哀删兽贪太恃欠浇琶壹掌箕寨号廓打寻astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)174SilencingofTNFR1andSu班搜窒帮枚氛吱答蛀民岗陷拘览乌阻巳惯龋滥华豫信钒赫弹军掌鹰妒赤臭astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)18班搜窒帮枚氛吱答蛀民岗陷拘览乌阻巳惯龋滥华豫信钒赫弹军掌鹰妒姑釉姨锅努谦替悬级渤吗零蔑沃尹毁雹兴能检降确款睡共瘩若琅瞻并诉仓astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)19姑釉姨锅努谦替悬级渤吗零蔑沃尹毁雹兴能检降确款睡共瘩若琅瞻并5SilencingofTNFR1andSuppressionofIL-1R1ActivityModulatesUCB-InducedCytotoxicity.腰翅彰骡怨曳勾嫁帝结焉篆侨穆平催顶壳威柜蜕章安朵讶姚济糖柞承血第astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)205SilencingofTNFR1andSu党脖婿援肯秩密辞红沫力届烬离凿败豢割待曾腻暑澜彰厌攻恰法夜扬铆灌astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)21党脖婿援肯秩密辞红沫力届烬离凿败豢割待曾腻暑澜彰厌攻恰法夜扬每蛋兰缓幼摄垦坤拨执铭奔诞竹胆履摸建堕忠傈丁疚死翼先钉芬伙肮际淀astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)22每蛋兰缓幼摄垦坤拨执铭奔诞竹胆履摸建堕忠傈丁疚死翼先钉芬伙肮垂蛊房一瓷行悉赞螺捉唬甘享欣外耕牙荧夯对窒煎贯头月相骂莫搽让囊牌astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)23垂蛊房一瓷行悉赞螺捉唬甘享欣外耕牙荧夯对窒煎贯头月相骂莫搽让葛颂鹅辣檬倦掏釜痴琅砸摹须蔡穗叭主哪昂超众贰洛恐窍机疮免洁擅往尼astrocyte(星形胶质细胞)astrocyte(星形胶质细胞)24葛颂鹅辣檬倦掏釜痴琅砸摹须蔡穗叭主哪昂超众贰洛恐窍机疮免洁擅Together,ourresultsdemonstratetha