astrocyte(星形膠質(zhì)細胞)

astrocyte(星形膠質(zhì)細胞)

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時間:2018-12-04

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1、AstrocyteReactivitytoUnconjugatedBilirubinRequiresTNF-αandIL-1βReceptorSignalingPathwaysGLIA59:14–25(2011)SCI(2010):5.19周康康2012-7-211Wehavereportedthattumornecrosisfactor(TNF)-αandinterleukin(IL)-1βareproducedbyculturedneuronsandmainlybyglialcellsexposedtounconjugatedbilirubin(UCB).The

2、effectsofthesecytokinesaremediatedbycellsurfacereceptorsthroughanuclearfactor(NF)-κB-dependentpathwaythatwehaveshowedtobeactivatedbyUCB.Summary22ExposureofastrocytestoUCBincreasedtheexpressionofbothTNF-αreceptorTNFR1andIL-1βreceptorIL-1R1,butnotTNFR2,aswellastheiractivation,observedbya

3、ugmentedbindingofreceptors’molecularadaptors,TRAF2andTRAF6,respectively.3SilencingofTNFR1,usingsiRNAtechnology,orblockadeofIL-1βcascade,usingitsendogenousantagonist,IL-1receptorantagonist(IL-1ra),preventedUCB-inducedcytokinereleaseandNF-κBactivation.34Interestingly,lackofTNF-αsignaltra

4、nsductionreducedUCB-inducedcelldeathforshortperiodsofincubation,incontrast,inhibitionofIL-1βcascadeproducedasustainedblockadeofastrocyteinjurybyUCB.5Together,ourdatashowthatinflammatorypathwaysareactivatedduringinvitroexposureofratastrocytestoUCB.Thissupportstheconceptthatinflammatoryp

5、athwaysplayaroleinbraindamagebyUCB,andthattheymayrepresentimportantpharmacologicaltargets.4Materialsandmethods1PrimaryCultureofAstrocytes:2-day-oldWistarrats2TransientTransfection:threedifferentdoublestrandedratTNFR1smallinterfering(si)RNAs(30nM),scrambledsiRNA(negativecontrol)ortheabs

6、enceofsiRNA(mockcontrol).3CellTreatment:50μMUCBplus100μMhumanserumalbumin(HSA)(UCBtoHSAmolarratioof0.5),from15minto24h,at370C.4WesternBlot:TheproteinexpressionofTNFR1,TNFR2,andIL-1R1weredeterminedbyWesternblotanalysis.55MeasurementofCytokineRelease:TNF-α,IL-1β,andIL-6withspecificDuoSet

7、RELISADevelopmentkits.6DetectionofNF-κBActivation—immunofluorescencedetection:rabbitanti-p65NF-κBsubunitantibody(1:200)astheprimaryantibodies,aFITC-labeledgoatanti-rabbitantibody(1:160)asthesecondaryantibodies.7EvaluationofCellDeath—LDHAstrocyteswerethenidentifiedinfixedcellsbyananti

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