资源描述：
《astrocyte(星形胶质细胞)》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。
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
