膜片钳原理技 术课件.ppt

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1、膜片钳原理技术膜片钳简介膜片钳记录方式膜片钳几种基本工作方式Cell-attachedrecordingCellPatch-pipetteCellmembraneExternallipidInternallipidWhole-cellrecordingCellPatch-pipetteCellmembraneInside-outrecordingPatch-pipetteTheinternalfaceofthelipidbi-layerfacesthebathsolutionOutside-outrecord

2、ingPatch-pipetteTheexternalfaceofthelipidbi-layerfacesthebathsolution膜片钳实验步骤膜片钳电流记录膜片钳的应用与细胞膜离子通道相关的药物作用药物相关离子通道治疗糖尿病药物钾离子通道抗心律失常药钠、钾钙通道心功能调节及保护钙、钾通道平滑肌调节钙、钾通道影响神经冲动、传导钠、钾、钙、氯通道免疫细胞（功能）调节钾、钙通道降压药钙通道β-淀粉样肽对海马神经元钾通道的影响Fig.Effectsofβ-AP25-353μmol.L-1onthecultu

3、redrathippocampalneurons.CellsviabilitywasevaluatedbytheMTTreductionassay.Valuesarethemean±SEMofthreeseparateexperimentsperformentsintriplicate.Fig.Delayedrectiferingpotassiumcurrent(IK)inculturedrathippocampalneurons.（A）IKrecordedfromnormalneurons;（B）IKrec

4、ordedfromneuronsafterexposedto3μmol.L-1beta-amyloid-peptide25-35for24h;（C）IKrecordedfromneuronsafterexposedto10μmol.L-1beta-amyloid-peptide25-35for12h.Kv2.1通道的稳定转染和高表达系统的建立Fig.HEK293cellsweretransientlytransfectedwithpEGFP-N1andpCR/CMVKv2.1.Eachofpanelswere

5、capturedbyCCDunderfluorescencemicroscopytovisualizeGFP.(left100X,right400X)2nA100msFig.RepresentativecurrentofKv2.1channelinstabletransfectedHEK293cellline.Whole–cellscurrentsevokedduring250msdepolarizingvoltagesteps10mvtorestpotentialsbetween–50and+40mVfro

6、maholdingpotentialof–50mV.kv2.1Fig.PropertiesofsteadystateactivationofKv2.1channelinstabletransfectedHEK293cell.Whole–cellscurrentsevokedduring250msdepolarizingvoltagestepstorestpotentialsbetween–50and+40mVfromaholdingpotentialof–50mV.Fig.Effectsof10µMgalan

7、tamineonI-Vrelationship(B)andtheactivationcurve(C)ofIK(DR)andIK(A).In(B),eachpointrepresentsmeanSEMof7cellsforIK(DR)and6cellsforIK(A).*P<0.05,**P<0.01vscontrol.In(C),galantaminecauseda4mVhyperpolarizingshiftoftheactivationcurveofIK(DR)(n=9),andnoeffectonth

8、atofIK(A)(n=6).Fig.Effectof10µMrivastigmineonthevoltagedependenceofinactivationofIK(A)andIK(DR).(A)Theoriginalcurrenttracesbeforeandaftertheadditionof10µMrivastigmine.(B)Rivastigminecausedbothhyperpola