膜片鉗原理技-術(shù)課件

膜片鉗原理技術(shù)1概況一點擊此處輸入相關(guān)文本內(nèi)容點擊此處輸入相關(guān)文本內(nèi)容整體概述概況三點擊此處輸入相關(guān)文本內(nèi)容點擊此處輸入相關(guān)文本內(nèi)容概況二點擊此處輸入相關(guān)文本內(nèi)容點擊此處輸入相關(guān)文本內(nèi)容2膜片鉗簡介3456膜片鉗記錄方式78膜片鉗幾種基本工作方式9Cell-attachedrecordingCellPatch-pipetteCellmembraneExternallipidInternallipid10Whole-cellrecordingCellPatch-pipetteCellmembrane11Inside-outrecordingPatch-pipetteTheinternalfaceofthelipidbi-layerfacesthebathsolution12Outside-outrecordingPatch-pipetteTheexternalfaceofthelipidbi-layerfacesthebathsolution1314膜片鉗實驗步驟15膜片鉗電流記錄16171819膜片鉗的應(yīng)用20與細胞膜離子通道相關(guān)的藥物作用藥物相關(guān)離子通道治療糖尿病藥物鉀離子通道抗心律失常藥鈉、鉀鈣通道心功能調(diào)節(jié)及保護鈣、鉀通道平滑肌調(diào)節(jié)鈣、鉀通道影響神經(jīng)沖動、傳導(dǎo)鈉、鉀、鈣、氯通道免疫細胞（功能）調(diào)節(jié)鉀、鈣通道降壓藥鈣通道21β-淀粉樣肽對海馬神經(jīng)元鉀通道的影響2223Fig.Effectsofβ-AP25-353μmol.L-1ontheculturedrathippocampalneurons.CellsviabilitywasevaluatedbytheMTTreductionassay.Valuesarethemean±SEMofthreeseparateexperimentsperformentsintriplicate.24Fig.Delayedrectiferingpotassiumcurrent(IK)inculturedrathippocampalneurons.（A）IKrecordedfromnormalneurons;（B）IKrecordedfromneuronsafterexposedto3μmol.L-1beta-amyloid-peptide25-35for24h;（C）IKrecordedfromneuronsafterexposedto10μmol.L-1beta-amyloid-peptide25-35for12h.25Kv2.1通道的穩(wěn)定轉(zhuǎn)染和高表達系統(tǒng)的建立26Fig.HEK293cellsweretransientlytransfectedwithpEGFP-N1andpCR/CMVKv2.1.EachofpanelswerecapturedbyCCDunderfluorescencemicroscopytovisualizeGFP.(left100X,right400X)27

2nA100msFig.RepresentativecurrentofKv2.1channelinstabletransfectedHEK293cellline.Whole–cellscurrentsevokedduring250msdepolarizingvoltagesteps10mvtorestpotentialsbetween–50and+40mVfromaholdingpotentialof–50mV.kv2.128Fig.PropertiesofsteadystateactivationofKv2.1channelinstabletransfectedHEK293cell.Whole–cellscurrentsevokedduring250msdepolarizingvoltagestepstorestpotentialsbetween–50and+40mVfromaholdingpotentialof–50mV.29Fig.Effectsof10μMgalantamineonI-Vrelationship(B)andtheactivationcurve(C)ofIK(DR)andIK(A).In(B),eachpointrepresentsmeanSEMof7cellsforIK(DR)and6cellsforIK(A).*P<0.05,**P<0.01vscontrol.In(C),galantaminecauseda4mVhyperpolarizingshiftoftheactivationcurveofIK(DR)(n=9),andnoeffectonthatofIK(A)(n=6).30Fig.Effectof10μMrivastigmineonthevoltagedependenceofinactivationofIK(A)andIK(DR).(A)Theoriginalcurrenttracesbeforeandaftertheadditionof10μMrivastigmine.(B)Rivastigminecausedbothhyperpolarizingshiftsofthesteady-stateinactivationofIK(A)andIK(DR)by11mVand27mV,respectively.n=5.31Ca2+3Na+Na+Na+

CellmodeltorecordoutwardcurrentofNa+-Ca2+exchanger膜片鉗全細胞記錄

雙向離子條件

高Na+電極內(nèi)液Na/Ca32雙向/生電性正向轉(zhuǎn)運SARCOLEMMAATPCa2+

Na+

Ca2+Ca2+channelCa2+ATPCa2+Sarcoplasmic

reticulumNCX33SARCOLEMMA2K+3Na+Na+H+3Na+NCXCa2+ATPNCXATPH+心肌缺血/再灌34SARCOLEMMA2K+3Na+Na+H+3Na+NCXCa2+Ca2+overload心肌缺血誘發(fā)反向轉(zhuǎn)運ATPNCXNa+35正常Na+-Ca2+交換電流在豚鼠心室肌細胞上的記錄+60mV-40mV-100mVA:currentbeforeNi2+5mMB:currentafterNi2+5mMA-B:Na+-Ca2+exchangecurrent(INa-Ca).5mMNi2+36胞外Ca2+濃度為1.8mM電極內(nèi)液中Na+濃度為25mM5mMNi2+3min左右達到最大持續(xù)5min未見“衰減”(run-down)現(xiàn)象37細胞內(nèi)Na+濃度對Na+-Ca2+交換電流的作用Currentdensities(pA/pF)（JournalofCardiovascularPharmacology2002）38Amiloride對豚鼠心室肌細胞Na+-Ca2+交換電流的作用Currentdensities(pA/pF)Amiloride39Currentdensities(pA/pF)KB-R7943KB-R7943對豚鼠心室肌細胞Na+-Ca2+交換電流的作用（JournalofCardiovascularPharmacol