膜片钳原理技 术

1、膜片钳原理技术膜膜 片片 钳钳 简简 介介膜膜 片片 钳钳 记记 录录 方方 式式膜片钳几种基本工作方式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膜片钳实验步骤膜膜 片片 钳钳 电电 流流 记记 录录膜膜 片片 钳钳 的的 应应 用用与细胞膜离子通道相关的药物作用与细胞膜离子通道相关的药物作用药物药物 相关离子通道相关离子通道治疗糖尿病药物治疗糖尿病药物 钾离子通道钾离子通道抗心律失常药抗心律失常药 钠、钾钙通道钠、钾钙通道心功能调节及保护心功能调节及保护 钙、钾通道钙、钾通道平滑肌调节平滑肌调节 钙、钾通道钙、钾通道影响神经冲动、传导影响神经冲动、传导 钠、钾、钙、氯通道钠、钾、钙、氯通道

3、免疫细胞（功能）调节免疫细胞（功能）调节 钾、钙通道钾、钙通道降压药降压药 钙通道钙通道-淀粉样肽对海马神经元钾通道的影响淀粉样肽对海马神经元钾通道的影响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通道的稳定转染和高表达系统的建立通道的稳定转染和高表达系统的建立 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+ 双向双向/ /生电性生电性正正 向向 转转 运运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心肌缺血诱发反向转运心肌缺血诱发反向转运ATPNa+正常正常Na+-Ca2+交换电流在豚鼠心室肌细胞上的记录交换电流在豚鼠心室肌细胞上的记录+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+浓度为浓度为1.8 mM电极内液中电极内液中Na+浓度为浓度为25 mM 5 mM Ni2+3 min左右达到最大左右达到最大持续持续5 min 未见未见“衰减衰减”(run-down)现现象象细胞内细胞内Na+浓度对浓度对Na+-Ca2+交换电流的作用交换电流的作用 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对豚鼠心室肌细胞对豚鼠心室肌细胞Na+-Ca2+交换电流的作用交换电流的作用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对豚鼠心室肌细胞对豚鼠心室肌细胞Na+-Ca2+交换电流的作用交换电流的作用（Journal of Cardiovascular PharmacologySZA对豚鼠心室肌细胞对豚鼠心室肌细胞Na+-Ca2+交换电流的作用交换电流的作用 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)膜片钳放大器的工作原理膜片钳放大器的工作原理膜片钳基本电路原理

13、膜片钳基本电路原理细胞等效电路基本电生理测量-伏安放大器n电压检测n电流检测电压钳电压钳单电极电压钳单电极膜片钳膜片钳实验装置膜片钳实验装置膜片钳实验系统配置单细胞膜片钳实验配置1, 膜片钳放大器 (Patch clamp amplifier)2, 显微操作仪（Micromanipulator）3, 微电极拉制仪（Pipette Puller）4, 打磨抛光仪（Microforge or Beveler）5, 灌流系统（Perfusion System）6, 细胞灌流槽（Chamber)6, 显微镜（Microscope）7, 摄像头和监视器（Camera , Monitor）8, 防震台和静电屏蔽网（Antivibration and Cage）9, 电脑和打印机 （Computer, Print