小鼠心室肌细胞分离方法的改良及钾电流的记录

目的报道一种改良的小鼠心室肌细胞分离方法,并观察小鼠心室肌细胞动作电位以及钾电流的电生理特性。方法采用双酶消化法分离单个心室肌细胞,应用全细胞膜片钳技术记录动作电位和钾电流。先记录外向钾电流(Ipeak),用低浓度4-氨基吡啶(100μmol/L)使延迟整流钾电流(IKur)失活后记录瞬时外向钾电流(Ito),用Ipeak减去Ito即可得到IKur,在完全失活IKur及Ito后可记录到稳态钾电流(Iss)。结果本法分离所得心室肌细胞横纹清晰,具有正常电生理活性,细胞池中加入层粘连蛋白后有助于细胞贴壁,从而易于形成高阻封接,并记录出小鼠心室肌细胞特征性的动作电位和钾电流。结论本实验所采用的分离方法简便,获得的小鼠心室肌细胞易于封接,且具有正常的电生理活性。

An improved method for mouse ventricular myocardium isolation and the recording of potassium currents

Objective To improve the method used to isolate adult mouse ventricular myocytes and observe the charac- teristics of action potential and potassium currents of myocytes. Methods Double enzymes digestion method was used to isolate single ventricular myocardium and whole-cell patch clamp recording technique was used to record AP and K＋ currents. Record Ipeak first, then use light concentration 4-AP（ 100 μmol/L）to devitalize IKur and record I10, so IKur = Ipeak -- Ito. And Iss can be record after the inactivation of Ikur and I10. Results Clear transverse striation could be observed in the cells of mouse ventricular myocardium which were separated by this way. These cells also had normal electrophysiology activity. Adding laminin in the cell pool contributes to cell adhesion, and Giga-ohm seal could be easily achieved. The characteristic action potential and potassium current in the mouse ventricular myocardium could be recorded. Conclusions The way to separate mouse ventricular myocardium in this experiment is very convenient, and the cells not only are very easy to seal, but also have normal electrophysiology activity.