豚鼠心肌细胞分离方法及电生理特性的观察

Isolation of the ventricular myocytes and observation of its electrophysiological properties in guinea-pig

目的:建立膜片钳实验室单个心肌细胞的分离方法和膜片钳技术平台。方法:采用酶解法分离豚鼠心室肌细胞;在膜片钳全细胞模式下记录不同基础刺激周长时的动作电位以及L-型钙电流、延迟性整流钾电流。结果:酶解法分离豚鼠心室肌可以得到90%的存活细胞;豚鼠的单个心室肌细胞静息电位为(-74.0±2.2)mV,基础刺激周长1000ms时复极90%的动作电位时程(APD90)为(313.2±20.72)ms;APD90和复极50%的动作电位时程(APD50)均随着基础刺激周长的延长而延长。L-型钙电流峰值电流密度为(-7.36±1.10)pA/pF,0.1mmol/Lverapamil灌流后为(-1.22±0.49)pA/pF,100μmol/LCdCl2灌流后内向电流消失;延迟性整流钾电流密度为(4.88±0.96)pA/pF,2μmol/L Dofetilide灌流后降低为(3.48±0.37)pA/pF。结论:酶解法分离的豚鼠心室肌细胞可以满足膜片钳实验的要求,此膜片钳技术可作为更深入电生理研究的平台。

Objective： To investigate the method of the guinea-pig ventricular myocytes and setup the stage for patch-clamping technique. Method： Single myocytes were isolated enzymatically from the guinea-pig ventricle. It was recorded that the action potential of different basic circle length, ICa-L and IK of ventricular myocytes in W-C Mode. Results： The isolated myocytes were survived by 90 % with enzymatic dissociation; the resting potential of single myocytes was -74.0±2.2mV, APDg0 313.2±20.72 ms when BCL was 1 000 ms; the peak of ICa-L current density -7.36±1.10 pA/pF, -1.22±0.49 pA/pF after perfusing with 0.1 mmol/L verapamil respectively, and the inward current was eliminated with perfusion of 100 μmol/L CdCl2 ; IK was 4.88± 0.96 pA/pF, 3.48 ± 0.37 pA/pF after 2μmol/L Dofetilide respectively. Conclusion： The single ventricular myocytes isolated enzymatically can be applied for patch-clamping experiments successfully; the patch-clamping technique can service for advanced electrophysiological research.