适于膜片钳实验的心肌细胞分离方法及维拉帕米对钙电流的作用

目的：建立适应于膜片钳实验的大鼠单个心室肌细胞的分离方法,探讨L-型钙通道在大鼠心肌电活动中的作用.方法：采用改进Langendorff装置,酶解法分离单个心室肌细胞,应用全细胞膜片钳技术记录心室肌细胞L-型钙电流及维拉帕米的作用.结果：该法分离到的心肌细胞存活率达80％～90％,高倍镜下细胞呈杆状,边缘清楚,表面光滑,纹理清晰;成功记录到典型L-型钙电流（Ica-L）,给予维拉帕米（0.3、3、30 μmol/L）后呈浓度依赖性抑制钙电流,其抑制率分别为（44.35±5.52）％,（61.06±2.99）％,（71.88±4.45）％.结论：改进心肌细胞的分离方法可获得大量耐钙心室肌细胞,这些细胞具有正常的电生理特性.

Improvement of Cardiac Myocytes Isolation Method for Patch Clamp Experiment and the Effect of Verapmil on Calcium Current

Objective：To establish a method for isolating single cardiac myocyte for patch clamp experiment,and to explore the role of L-type calcium channels in cardiac electrical activity in rats.Methods：The ventricular myocytes were isolated by using enzymolysis method with improved Langendorff apparatus.L-type calcium current and the effect of verapmil were recorded by using whole-cell patch clamp technique.Results：The survival rate of myocardial cells was 80％ ～90％.The cells were rod-shaped with clear edge,smooth surface,clear texture at high magnification.L-type calcium current was recorded successfully.Verapamil inhibited the L-type calcium current in a concentrationdependent manner.The inhibitory rate of 0.3,3,30 μmol/L verapamil were （44.35 ± 5.52） ％,（61.06 ± 2.99） ％,（71.88 ± 4.45） ％ respectively.Conclusions：The improved isolating method can obtain a large number of single cardiac myocytes with strong calcium resistance ability and normal electrophysiological properties.