1、膜片鉗原理技術膜膜 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膜片鉗實驗步驟膜膜 片片 鉗鉗 電電 流流 記記 錄錄膜膜 片片 鉗鉗 的的 應應 用用與細胞膜離子通道相關的藥物作用與細胞膜離子通道相關的藥物作用藥物藥物 相關離子通道相關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、鉀鈣通道心功能調節及保護心功能調節及保護 鈣、鉀通道鈣、鉀通道平滑肌調節平滑肌調節 鈣、鉀通道鈣、鉀通道影響神經沖動、傳導影響神經沖動、傳導 鈉、鉀、鈣、氯通道鈉、鉀、鈣、氯通道

3、免疫細胞（功能）調節免疫細胞（功能）調節 鉀、鈣通道鉀、鈣通道降壓藥降壓藥 鈣通道鈣通道-淀粉樣肽對海馬神經元鉀通道的影響淀粉樣肽對海馬神經元鉀通道的影響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通道的穩定轉染和高表達系統的建立通道的穩定轉染和高表達系統的建立 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 轉轉 運運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心肌缺血誘發反向轉運心肌缺血誘發反向轉運ATPNa+正常正常Na+-Ca2+交換電流在豚鼠心室肌細胞上的記錄交換電流在豚鼠心室肌細胞上的記錄+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電極內液中電極內液中Na+濃度為濃度為25 mM 5 mM Ni2+3 min左右達到最大左右達到最大持續持續5 min 未見未見“衰減衰減”(run-down)現現象象細胞內細胞內Na+濃度對濃度對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對豚鼠心室肌細胞對豚鼠心室肌細胞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對豚鼠心室肌細胞對豚鼠心室肌細胞Na+-Ca2+交換電流的作用交換電流的作用（Journal of Cardiovascular PharmacologySZA對豚鼠心室肌細胞對豚鼠心室肌細胞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胞等效電路基本電生理測量-伏安放大器n電壓檢測n電流檢測電壓鉗電壓鉗單電極電壓鉗單電極膜片鉗膜片鉗實驗裝置膜片鉗實驗裝置膜片鉗實驗系統配置單細胞膜片鉗實驗配置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（Perfusion System）6, 細胞灌流槽（Chamber)6, 顯微鏡（Microscope）7, 攝像頭和監視器（Camera , Monitor）8, 防震臺和靜電屏蔽網（Antivibration and Cage）9, 電腦和打印機 （Computer, Print