excadp调控大鼠脊髓星形胶质细胞ampk活性参与神经病理性疼痛的作用及机制

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1、第三军医大学博士学位论文rpmRoundperminute每分钟转数RLUrelativeluminescenceunit相对荧光单位sSecond秒STAT3Signaltransducer，andactivatorof信号转导子和转录激活子3transcription3Tristhrihydroxyaminomethane三羟甲基氨基甲烷UUnit单位3第三军医大学博士学位论文EffectofextracellularadenosinediphosphateonneuropathicpainviaAMPK

2、inspinalastrocytesAbstractGlialcellsactivationplaysimportantroleinthemaintenanceandpromotionofneuropathicpain(NPP).ExtracellularATPisapowerfulstimulatortoactivateglialcells,butitsoriginsandtheproducingmechanismsareunclear.EarlyexperimentalresultssuggestedP2

3、YreceptorshaveeffectsonthemodulationoftheintracellularATPlevel,andnowneedtodetecttheireffectsonextracellularATPlevelandfindoutthemechanisms.AMPKisthekeyregulatorofATPsynthesisandexpenditure,itsupstreamsignalsissometimesconsistencywiththedownstreamsignalsofG

4、-protein-coupledP2Yreceptors.WhetherAMPKisamodulatorofthesignalsfromP2Yreceptorstomitochondrionneedtobestudied.ThistopicaimstodetecttheeffectsofAMPKontheratspinaldorsalhornastrocytes.Experimentaltechniquessuchasmorphology,MTTassay,Westernblotandhighperforma

5、nceliquidchromatographywereemployedtostudytheeffectsofextracellularADPonATPsynthesis,releaseandAMPKexpressioninastrocytesinvitro.EffectsofAMPKinhibitor(CompoundC)onthebehaviorofpainandGFAPexpressionwereobservedinchroniccontractedsciaticnerveinjury(CCI)ratst

6、ostudytherelationshipaboutAMPK,astrocyteactivationandchronicpainoccur.ResearchresultswillbehelpfulforunderstandingthemechanismsofATP-inducedpainandfindinganewwaytoinhibitNPP.Methods:PrimarydissociatedspinaldorsalhornastrocyteswerepreparedfromSprague–Dawleyr

7、atsforstudyingtheeffectsofADPoncellsinvitro.CellularATPlevelsweremeasuredusingafireflyluciferaseATPassaykitandmitochondrialmembranepotential(MMP)wasassessedbyusing5,5,6,6-tetrachloro-1,1’3,3’-tetraethylbenzamidazol-carboncyanine(JC-1)inbothadirectandanindir

8、ectmanner.ExpressionofAMPKintheastrocytesweredeterminedbymuti-labelingimmunofluorescenceandwesternblotmethods.EffectofCompoundConADPandADPβS-evokedglutamatereleaseweredetectedbyusingHPLC.Theratswithint