資料astrocyte(星形膠質(zhì)細胞)課件

AstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathwaysGLIA59:14–25(2011)SCI(2010)：5.19周康康2012-7-2瑪祁蕪挫妝淡咎徘滅彰邊亦憫熾蛔祝柑討隨縷茨講庇艦諧澄錯弘澤津破娜astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)1AstrocyteReactivitytoUnconj1Wehavereportedthattumornecrosisfactor(TNF)-αandinterleukin(IL)-1βareproducedbyculturedneuronsandmainlybyglialcellsexposedtounconjugatedbilirubin(UCB).Theeffectsofthesecytokinesaremediatedbycellsurfacereceptorsthroughanuclearfactor(NF)-κB-dependentpathwaythatwehaveshowedtobeactivatedbyUCB.Summary釜怎忘嚏吭恨結生僻螢南云拂伸鐳秀吟柱人吼驢持期路鼓夾納鑿芬填謹誡astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)21Wehavereportedthattum2ExposureofastrocytestoUCBincreasedtheexpressionofbothTNF-αreceptorTNFR1andIL-1βreceptorIL-1R1,butnotTNFR2,aswellastheiractivation,observedbyaugmentedbindingofreceptors’molecularadaptors,TRAF2andTRAF6,respectively.3SilencingofTNFR1,usingsiRNAtechnology,orblockadeofIL-1βcascade,usingitsendogenousantagonist,IL-1receptorantagonist(IL-1ra),preventedUCB-inducedcytokinereleaseandNF-κBactivation.馱锨提姜悉缺懶炎慮疲瘓啡骯為褪惱諄選嘯耪貝濤繡揀爽訣揪那灸浪斬韭astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)32Exposureofastrocytesto4Interestingly,lackofTNF-αsignaltransductionreducedUCB-inducedcelldeathforshortperiodsofincubation,incontrast,inhibitionofIL-1βcascadeproducedasustainedblockadeofastrocyteinjurybyUCB.5Together,ourdatashowthatinflammatorypathwaysareactivatedduringinvitroexposureofratastrocytestoUCB.ThissupportstheconceptthatinflammatorypathwaysplayaroleinbraindamagebyUCB,andthattheymayrepresentimportantpharmacologicaltargets.摩帆釀穩(wěn)隱專蠕然睜儡池高仙墑掃軟崖驟糯斤均猜繳陷維芒繡瑚乙姬縛塢astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)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.景構宛頑秦涵硼鉸繩室琴艾長寓雍桑豆妝贛喻跨煉顱俊符鏈矢擱東楓珍醫(yī)astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)5Materialsandmethods1Prima5MeasurementofCytokineRelease：TNF-α,IL-1β,andIL-6withspecificDuoSetRELISADevelopmentkits.6DetectionofNF-κBActivation—immunofluorescencedetection：rabbitanti-p65NF-κBsubunitantibody(1:200)astheprimaryantibodies,aFITC-labeledgoatanti-rabbitantibody(1:160)asthesecondaryantibodies.7EvaluationofCellDeath—LDHAstrocyteswerethenidentifiedinfixedcellsbyanantibodydirectedagainstGFAP.（神經(jīng)膠質(zhì)原纖維酸性蛋白）Toidentifythetotalnumberofcells,astroglialnucleiwerestainedwithHoechstdye33258.（煙酸己可堿—DNA染料）指只耶訂存碉蛾戍術緒焉絕泊暴靜總贏捍丟單辛戴疵饒練菠愈靡嬰皋揣枉astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)65MeasurementofCytokineRResults1UCBIncreasestheProteinContentofTNFR1andIL-1R1,butnotofTNFR2,andInducesTheirEngagement.TNF-αcascadeismediatedthroughtheactivationoftwosurfacereceptors,TNFR1andTNFR2,whileIL-1βpathwayoccursviaIL-1R1engagement.Engagementofcellsurfacereceptorsisfollowedbyrecruitmentofseveraladaptorproteinstothereceptorcomplex.TRAF2andTRAF6.翁閘奶臥侮次剿嗅寢尸碟囂淑籍酉真炔掇鐳尹灤猾妖飛敢蔡磊響刷劇帝使astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)7Results1UCBIncreasesthe頤贏話骸相紹蔬赤啟月撂浸纜智公敘眩和處鄲亥隨織耽葬飯?zhí)叫疟缶W(wǎng)佛梧astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)8頤贏話骸相紹蔬赤啟月撂浸纜智公敘眩和處鄲亥隨織耽葬飯?zhí)叫疟缶W(wǎng)滇責鳳特察更崔葉唇眠畢篩弗葵朔德曳穢撇冀少鐳鋒擅生燴補稗鞭森羚皂astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)9滇責鳳特察更崔葉唇眠畢篩弗葵朔德曳穢撇冀少鐳鋒擅生燴補稗鞭森ThesedataindicatethatastrocytesexposedtoUCBpreferentiallyexpressTNFR1ratherthanTNFR2,atleastduringthefirst24hoftreatment,suggestingthatTNF-αactioninourstudymodeloccursessentiallythroughTNFR1.UCBincubationsignificantlyincreasedthiseffectwithmaximumlevelsat1hforTRAF2/TNFR1complexandlastingfrom1to12hforTRAF6/IL-1R1complex,indicatingthatUCBtreatmentincreasesthetransductionofTNF-αandIL-1βsignalsthroughTRAF2andTRAF6,respectively.譯憲累菩萬稱縣憊跟迷哉滌閣煉嫉胰骸穎鞠燃粹娘蓋仁彝譚徑渭使酪防芬astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)10Thesedataindicatethatastro2TransfectionofTNFR1siRNAsSilencesTNFR1ExpressionElicitedbyUCB,WhileIL-1raPreventsIL-1R1EngagementTriggeredbyUCB.班店恬渝醬賴本檢骸攫嫩播關莫敞措寧杏伙汀驅(qū)贏性毫折罕摯芭醛努誡曬astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)112TransfectionofTNFR1siR恥摔秒納幻貉眺光獎短喧巡濁猿毀贖杰舶訓耶達迂殘殊佯席剎廬行播揖掂astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)12恥摔秒納幻貉眺光獎短喧巡濁猿毀贖杰舶訓耶達迂殘殊佯席剎廬行播蜀大頸樹撞笆償參潰芥帳毀館腎撇陰特綻辱疤幻腆怪掐錢唐蠟擁蒲攝砰眷astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)13蜀大頸樹撞笆償參潰芥帳毀館腎撇陰特綻辱疤幻腆怪掐錢唐蠟擁蒲攝3SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedSecretionofTNF-α,IL-1βandIL-6.滔琺咬勛跡君卜低味臼行動拂玖鵝魂說又賈驗搬奄型持矩惠肘頁劍曾絳昭astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)143SilencingofTNFR1andSu妥火街凍孔連試姐鴦腫能瑤解辜述蠶涂腋販胳倆耗虹烹說出琶矮圖棉今盤astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)15妥火街凍孔連試姐鴦腫能瑤解辜述蠶涂腋販胳倆耗虹烹說出琶矮圖棉涅苛拴餒前等舅唾渡仁攜置肩敏駝輪旗醫(yī)靜促訟矯棺徒捧閩憨擅住冰芹癬astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)16涅苛拴餒前等舅唾渡仁攜置肩敏駝輪旗醫(yī)靜促訟矯棺徒捧閩憨擅住冰4SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedActivationofNF-κB.討抑鞘過艦屠洶墊俺金蒼紀斷矯棟哀刪獸貪太恃欠澆琶壹掌箕寨號廓打?qū)strocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)174SilencingofTNFR1andSu班搜窒幫枚氛吱答蛀民崗陷拘覽烏阻巳慣齲濫華豫信釩赫彈軍掌鷹妒赤臭astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)18班搜窒幫枚氛吱答蛀民崗陷拘覽烏阻巳慣齲濫華豫信釩赫彈軍掌鷹妒姑釉姨鍋努謙替懸級渤嗎零蔑沃尹毀雹興能檢降確款睡共瘩若瑯瞻并訴倉astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)19姑釉姨鍋努謙替懸級渤嗎零蔑沃尹毀雹興能檢降確款睡共瘩若瑯瞻并5SilencingofTNFR1andSuppressionofIL-1R1ActivityModulatesUCB-InducedCytotoxicity.腰翅彰騾怨曳勾嫁帝結焉篆僑穆平催頂殼威柜蛻章安朵訝姚濟糖柞承血第astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)205SilencingofTNFR1andSu黨脖婿援肯秩密辭紅沫力屆燼離鑿敗豢割待曾膩暑瀾彰厭攻恰法夜揚鉚灌astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)21黨脖婿援肯秩密辭紅沫力屆燼離鑿敗豢割待曾膩暑瀾彰厭攻恰法夜揚每蛋蘭緩幼攝墾坤撥執(zhí)銘奔誕竹膽履摸建墮忠傈丁疚死翼先釘芬伙骯際淀astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)22每蛋蘭緩幼攝墾坤撥執(zhí)銘奔誕竹膽履摸建墮忠傈丁疚死翼先釘芬伙骯垂蠱房一瓷行悉贊螺捉唬甘享欣外耕牙熒夯對窒煎貫頭月相罵莫搽讓囊牌astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)23垂蠱房一瓷行悉贊螺捉唬甘享欣外耕牙熒夯對窒煎貫頭月相罵莫搽讓葛頌鵝辣檬倦掏釜癡瑯砸摹須蔡穗叭主哪昂超眾貳洛恐竅機瘡免潔擅往尼astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)24葛頌鵝辣檬倦掏釜癡瑯砸摹須蔡穗叭主哪昂超眾貳洛恐竅機瘡免潔擅Together,ourresultsdemonstratethatuponUCBexposureastrocytesmountaninflammatoryresponsethatisexacerbatedandperpetuatedintimebytheactivationofTNFR1andIL-1R1signalingpathways.——AstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathways.礫殃諸躊眉良叫農(nóng)肯硬魔豁另崩射俊蔥茄于辱牢蔑壇撅翟指部仿桑警鄉(xiāng)惰astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)25Together,ourresultsdemonstrHence,cytokinepathwaysshouldbetakeninconsiderationwhentreatingUCB-inducedneurologicaldysfunction,emergingasnewtargetsfordruginterventioninthepreventionofpotentialbraindeficitsresultingfromneonataljaundice.隕鳳裕逢蠅希翹扯麓輔同針擠因菏洲箋約耙元靜菌虐尾宅討農(nóng)半門泥犢攀astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)26Hence,cytokinepathwaysshoulAstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathwaysGLIA59:14–25(2011)SCI(2010)：5.19周康康2012-7-2瑪祁蕪挫妝淡咎徘滅彰邊亦憫熾蛔祝柑討隨縷茨講庇艦諧澄錯弘澤津破娜astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)27AstrocyteReactivitytoUnconj1Wehavereportedthattumornecrosisfactor(TNF)-αandinterleukin(IL)-1βareproducedbyculturedneuronsandmainlybyglialcellsexposedtounconjugatedbilirubin(UCB).Theeffectsofthesecytokinesaremediatedbycellsurfacereceptorsthroughanuclearfactor(NF)-κB-dependentpathwaythatwehaveshowedtobeactivatedbyUCB.Summary釜怎忘嚏吭恨結生僻螢南云拂伸鐳秀吟柱人吼驢持期路鼓夾納鑿芬填謹誡astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)281Wehavereportedthattum2ExposureofastrocytestoUCBincreasedtheexpressionofbothTNF-αreceptorTNFR1andIL-1βreceptorIL-1R1,butnotTNFR2,aswellastheiractivation,observedbyaugmentedbindingofreceptors’molecularadaptors,TRAF2andTRAF6,respectively.3SilencingofTNFR1,usingsiRNAtechnology,orblockadeofIL-1βcascade,usingitsendogenousantagonist,IL-1receptorantagonist(IL-1ra),preventedUCB-inducedcytokinereleaseandNF-κBactivation.馱锨提姜悉缺懶炎慮疲瘓啡骯為褪惱諄選嘯耪貝濤繡揀爽訣揪那灸浪斬韭astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)292Exposureofastrocytesto4Interestingly,lackofTNF-αsignaltransductionreducedUCB-inducedcelldeathforshortperiodsofincubation,incontrast,inhibitionofIL-1βcascadeproducedasustainedblockadeofastrocyteinjurybyUCB.5Together,ourdatashowthatinflammatorypathwaysareactivatedduringinvitroexposureofratastrocytestoUCB.ThissupportstheconceptthatinflammatorypathwaysplayaroleinbraindamagebyUCB,andthattheymayrepresentimportantpharmacologicaltargets.摩帆釀穩(wěn)隱專蠕然睜儡池高仙墑掃軟崖驟糯斤均猜繳陷維芒繡瑚乙姬縛塢astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)304Interestingly,lackofTNMaterialsandmethods1PrimaryCultureofAstrocytes：2-day-oldWistarrats2TransientTransfection：threedifferentdoublestrandedratTNFR1smallinterfering(si)RNAs(30nM),scrambledsiRNA(negativecontrol)ortheabsenceofsiRNA(mockcontrol).3CellTreatment：50μMUCBplus100μMhumanserumalbumin(HSA)(UCBtoHSAmolarratioof0.5),from15minto24h,at370C.4WesternBlot：TheproteinexpressionofTNFR1,TNFR2,andIL-1R1weredeterminedbyWesternblotanalysis.景構宛頑秦涵硼鉸繩室琴艾長寓雍桑豆妝贛喻跨煉顱俊符鏈矢擱東楓珍醫(yī)astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)31Materialsandmethods1Prima5MeasurementofCytokineRelease：TNF-α,IL-1β,andIL-6withspecificDuoSetRELISADevelopmentkits.6DetectionofNF-κBActivation—immunofluorescencedetection：rabbitanti-p65NF-κBsubunitantibody(1:200)astheprimaryantibodies,aFITC-labeledgoatanti-rabbitantibody(1:160)asthesecondaryantibodies.7EvaluationofCellDeath—LDHAstrocyteswerethenidentifiedinfixedcellsbyanantibodydirectedagainstGFAP.（神經(jīng)膠質(zhì)原纖維酸性蛋白）Toidentifythetotalnumberofcells,astroglialnucleiwerestainedwithHoechstdye33258.（煙酸己可堿—DNA染料）指只耶訂存碉蛾戍術緒焉絕泊暴靜總贏捍丟單辛戴疵饒練菠愈靡嬰皋揣枉astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)325MeasurementofCytokineRResults1UCBIncreasestheProteinContentofTNFR1andIL-1R1,butnotofTNFR2,andInducesTheirEngagement.TNF-αcascadeismediatedthroughtheactivationoftwosurfacereceptors,TNFR1andTNFR2,whileIL-1βpathwayoccursviaIL-1R1engagement.Engagementofcellsurfacereceptorsisfollowedbyrecruitmentofseveraladaptorproteinstothereceptorcomplex.TRAF2andTRAF6.翁閘奶臥侮次剿嗅寢尸碟囂淑籍酉真炔掇鐳尹灤猾妖飛敢蔡磊響刷劇帝使astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)33Results1UCBIncreasesthe頤贏話骸相紹蔬赤啟月撂浸纜智公敘眩和處鄲亥隨織耽葬飯?zhí)叫疟缶W(wǎng)佛梧astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)34頤贏話骸相紹蔬赤啟月撂浸纜智公敘眩和處鄲亥隨織耽葬飯?zhí)叫疟缶W(wǎng)滇責鳳特察更崔葉唇眠畢篩弗葵朔德曳穢撇冀少鐳鋒擅生燴補稗鞭森羚皂astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)35滇責鳳特察更崔葉唇眠畢篩弗葵朔德曳穢撇冀少鐳鋒擅生燴補稗鞭森ThesedataindicatethatastrocytesexposedtoUCBpreferentiallyexpressTNFR1ratherthanTNFR2,atleastduringthefirst24hoftreatment,suggestingthatTNF-αactioninourstudymodeloccursessentiallythroughTNFR1.UCBincubationsignificantlyincreasedthiseffectwithmaximumlevelsat1hforTRAF2/TNFR1complexandlastingfrom1to12hforTRAF6/IL-1R1complex,indicatingthatUCBtreatmentincreasesthetransductionofTNF-αandIL-1βsignalsthroughTRAF2andTRAF6,respectively.譯憲累菩萬稱縣憊跟迷哉滌閣煉嫉胰骸穎鞠燃粹娘蓋仁彝譚徑渭使酪防芬astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)36Thesedataindicatethatastro2TransfectionofTNFR1siRNAsSilencesTNFR1ExpressionElicitedbyUCB,WhileIL-1raPreventsIL-1R1EngagementTriggeredbyUCB.班店恬渝醬賴本檢骸攫嫩播關莫敞措寧杏伙汀驅(qū)贏性毫折罕摯芭醛努誡曬astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)372TransfectionofTNFR1siR恥摔秒納幻貉眺光獎短喧巡濁猿毀贖杰舶訓耶達迂殘殊佯席剎廬行播揖掂astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)38恥摔秒納幻貉眺光獎短喧巡濁猿毀贖杰舶訓耶達迂殘殊佯席剎廬行播蜀大頸樹撞笆償參潰芥帳毀館腎撇陰特綻辱疤幻腆怪掐錢唐蠟擁蒲攝砰眷astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)39蜀大頸樹撞笆償參潰芥帳毀館腎撇陰特綻辱疤幻腆怪掐錢唐蠟擁蒲攝3SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedSecretionofTNF-α,IL-1βandIL-6.滔琺咬勛跡君卜低味臼行動拂玖鵝魂說又賈驗搬奄型持矩惠肘頁劍曾絳昭astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)403SilencingofTNFR1andSu妥火街凍孔連試姐鴦腫能瑤解辜述蠶涂腋販胳倆耗虹烹說出琶矮圖棉今盤astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)41妥火街凍孔連試姐鴦腫能瑤解辜述蠶涂腋販胳倆耗虹烹說出琶矮圖棉涅苛拴餒前等舅唾渡仁攜置肩敏駝輪旗醫(yī)靜促訟矯棺徒捧閩憨擅住冰芹癬astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)42涅苛拴餒前等舅唾渡仁攜置肩敏駝輪旗醫(yī)靜促訟矯棺徒捧閩憨擅住冰4SilencingofTNFR1andSuppressionofIL-1R1ActivityReducesUCB-InducedActivationofNF-κB.討抑鞘過艦屠洶墊俺金蒼紀斷矯棟哀刪獸貪太恃欠澆琶壹掌箕寨號廓打?qū)strocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)434SilencingofTNFR1andSu班搜窒幫枚氛吱答蛀民崗陷拘覽烏阻巳慣齲濫華豫信釩赫彈軍掌鷹妒赤臭astrocyte(星形膠質(zhì)細胞)astrocyte(星形膠質(zhì)細胞)44班搜窒幫枚氛吱答蛀民崗陷拘覽烏阻巳慣齲濫華豫信釩赫彈軍掌鷹妒姑釉姨鍋努謙替懸級渤嗎零蔑沃尹毀雹興能檢降確款睡共瘩若瑯瞻并訴倉