膜片鉗原理技-術課件

膜片鉗原理技術1概況一點擊此處輸入相關文本內容點擊此處輸入相關文本內容整體概述概況三點擊此處輸入相關文本內容點擊此處輸入相關文本內容概況二點擊此處輸入相關文本內容點擊此處輸入相關文本內容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膜片鉗的應用20與細胞膜離子通道相關的藥物作用藥物相關離子通道治療糖尿病藥物鉀離子通道抗心律失常藥鈉、鉀鈣通道心功能調節(jié)及保護鈣、鉀通道平滑肌調節(jié)鈣、鉀通道影響神經(jīng)沖動、傳導鈉、鉀、鈣、氯通道免疫細胞（功能）調節(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+電極內液Na/Ca32雙向/生電性正向轉運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