膜片鉗原理技 術(shù)

1、膜片鉗原理技術(shù)膜膜 片片 鉗鉗 簡簡 介介膜膜 片片 鉗鉗 記記 錄錄 方方 式式膜片鉗幾種基本工作方式Cell-attached recordingCellPatch-pipetteCell membraneExternal lipidInternal lipidWhole-cell recordingCellPatch-pipetteCell membraneInside-out recordingPatch-pipetteThe internal face of the lipidbi-layer faces the bath solutionOutside-out recordingP

2、atch-pipetteThe external face of the lipidbi-layer faces the bath solution膜片鉗實(shí)驗(yàn)步驟膜膜 片片 鉗鉗 電電 流流 記記 錄錄膜膜 片片 鉗鉗 的的 應(yīng)應(yīng) 用用與細(xì)胞膜離子通道相關(guān)的藥物作用與細(xì)胞膜離子通道相關(guān)的藥物作用藥物藥物 相關(guān)離子通道相關(guān)離子通道治療糖尿病藥物治療糖尿病藥物 鉀離子通道鉀離子通道抗心律失常藥抗心律失常藥 鈉、鉀鈣通道鈉、鉀鈣通道心功能調(diào)節(jié)及保護(hù)心功能調(diào)節(jié)及保護(hù) 鈣、鉀通道鈣、鉀通道平滑肌調(diào)節(jié)平滑肌調(diào)節(jié) 鈣、鉀通道鈣、鉀通道影響神經(jīng)沖動(dòng)、傳導(dǎo)影響神經(jīng)沖動(dòng)、傳導(dǎo) 鈉、鉀、鈣、氯通道鈉、鉀、鈣、氯通道

3、免疫細(xì)胞（功能）調(diào)節(jié)免疫細(xì)胞（功能）調(diào)節(jié) 鉀、鈣通道鉀、鈣通道降壓藥降壓藥 鈣通道鈣通道-淀粉樣肽對(duì)海馬神經(jīng)元鉀通道的影響淀粉樣肽對(duì)海馬神經(jīng)元鉀通道的影響Fig . Effects of-AP25-35 3 mol.L-1 on the cultured rat hippocampal neurons . Cells viability was evaluated by the MTT reduction assay. Values are the mean SEM of three separate experiments performents in triplicate. +40 mV

4、-50 mV (A) (B) (C) 100 ms 1 nA Kv2.1通道的穩(wěn)定轉(zhuǎn)染和高表達(dá)系統(tǒng)的建立通道的穩(wěn)定轉(zhuǎn)染和高表達(dá)系統(tǒng)的建立 Fig. HEK293 cells were transiently transfected with pEGFP-N1 and pCR/CMV Kv2.1. Each of panels were captured by CCD under fluorescence microscopy to visualize GFP. (left 100X,right 400X) 2 nA100 msFig. Representative current of Kv2

5、.1 channel in stable transfected HEK293 cell line. Wholecells currents evoked during 250ms depolarizing voltage steps 10mv to rest potentials between 50 and +40mV from a holding potential of 50mV.kv2.1Fig. Properties of steady state activation of Kv2.1 channel in stable transfected HEK293 cell. Whol

6、ecells currents evoked during 250ms depolarizing voltage steps to rest potentials between 50 and +40mV from a holding potential of 50mV.Fig. Effects of 10 M g a l a n t a m i n e o n I - V relationship (B) and the activation curve (C) of IK(DR) and IK(A). In (B), each point represents mean SEM of 7

7、cells for IK(DR) and 6 cells for IK(A). *P 0.05, *P 0.01 vs control. In (C), galantamine caused a 4 mV hyperpolarizing shift of the activation curve of IK(DR) (n = 9), and no effect on that of IK(A) (n = 6). Fig. Effect of 10 M rivastigmine on the voltage dependence of inactivation of IK(A) and IK(D

8、R). (A) The original current traces before and after the addition of 10 M rivastigmine.(B) Rivastigmine caused both hyperpolarizing shifts of the steady-state inactivation of IK(A) and IK(DR) by 11 mV and 27 mV, respectively. n = 5.Ca2+3Na+Na+Na+ 雙向雙向/ /生電性生電性正正 向向 轉(zhuǎn)轉(zhuǎn) 運(yùn)運(yùn)SARCOLEMMACa2+ Na+ Ca2+Ca2+ c

9、hannelCa2+Ca2+Sarcoplasmic reticulumSARCOLEMMA2 K+Na+H+3 Na+NCXCa2+ATPH+心肌缺血心肌缺血/ /再灌再灌SARCOLEMMA2 K+Na+H+3 Na+NCXCa2+ Ca2+ overload心肌缺血誘發(fā)反向轉(zhuǎn)運(yùn)心肌缺血誘發(fā)反向轉(zhuǎn)運(yùn)ATPNa+正常正常Na+-Ca2+交換電流在豚鼠心室肌細(xì)胞上的記錄交換電流在豚鼠心室肌細(xì)胞上的記錄+60 mV-40 mV-100 mVA: current before Ni2+ 5 mM B: current after Ni2+ 5 mM A-B: Na+-Ca2+ exchange c

10、urrent (INa-Ca).5 mM Ni2+ 3 m i n 8 m i n 0 m i nNi 2+胞外胞外Ca2+濃度為濃度為1.8 mM電極內(nèi)液中電極內(nèi)液中Na+濃度為濃度為25 mM 5 mM Ni2+3 min左右達(dá)到最大左右達(dá)到最大持續(xù)持續(xù)5 min 未見未見“衰減衰減”(run-down)現(xiàn)現(xiàn)象象細(xì)胞內(nèi)細(xì)胞內(nèi)Na+濃度對(duì)濃度對(duì)Na+-Ca2+交換電流的作用交換電流的作用 Na+ 0 mM Na+ 15 mM Na+ 25 mM Na+ 35 mM12345678(min)Ni2+86420-2-4Current densities (pA/pF)（Journal of Ca

11、rdiovascular Pharmacology 2002）Amiloride對(duì)豚鼠心室肌細(xì)胞對(duì)豚鼠心室肌細(xì)胞Na+-Ca2+交換電流的作用交換電流的作用1 2 3 4 5 6 7 8 9 10 11(min)86420-2-4AmilorideNi2+ Amiloride 10 M Amiloride 30 M Amiloride 100 M Before drugCurrent densities (pA/pF)Amiloride1234567891011(min)86420-2-4KB-R7943Ni2+ KB-R7943 1 M KB-R7943 10 M Before drugCu

12、rrent densities (pA/pF)KB-R7943KB-R7943對(duì)豚鼠心室肌細(xì)胞對(duì)豚鼠心室肌細(xì)胞Na+-Ca2+交換電流的作用交換電流的作用（Journal of Cardiovascular PharmacologySZA對(duì)豚鼠心室肌細(xì)胞對(duì)豚鼠心室肌細(xì)胞Na+-Ca2+交換電流的作用交換電流的作用 1 2 3 4 5 6 7 8 9 10 11(min)86420-2-4SZANi2+ SZA 0.1 M SZA 1 M SZA 10 M Before drugSZACurrent densities (pA/pF)膜片鉗放大器的工作原理膜片鉗放大器的工作原理膜片鉗基本電路原理

13、膜片鉗基本電路原理細(xì)胞等效電路基本電生理測(cè)量-伏安放大器n電壓檢測(cè)n電流檢測(cè)電壓鉗電壓鉗單電極電壓鉗單電極膜片鉗膜片鉗實(shí)驗(yàn)裝置膜片鉗實(shí)驗(yàn)裝置膜片鉗實(shí)驗(yàn)系統(tǒng)配置單細(xì)胞膜片鉗實(shí)驗(yàn)配置1, 膜片鉗放大器 (Patch clamp amplifier)2, 顯微操作儀（Micromanipulator）3, 微電極拉制儀（Pipette Puller）4, 打磨拋光儀（Microforge or Beveler）5, 灌流系統(tǒng)（Perfusion System）6, 細(xì)胞灌流槽（Chamber)6, 顯微鏡（Microscope）7, 攝像頭和監(jiān)視器（Camera , Monitor）8, 防震臺(tái)和靜電屏蔽網(wǎng)（Antivibration and Cage）9, 電腦和打印機(jī) （Computer, Print