恒流灌注分离心肌细胞及膜片钳记录钾电流方法学探讨

目的探讨膜片钳实验中豚鼠、大鼠心肌细胞急性分离过程中的观察指标和影响因素,并进行膜片钳记录。方法使用改进的Langendorff恒流灌注法分离心肌细胞和全细胞膜片钳记录钾通道电流。结果分离即刻,存活细胞可达80%以上,复钙后约40%～50%呈静息状态,余出现自发性收缩,存活细胞能够进行膜片钳封接,于室温KB液中可成活8 h以上。结论使用恒流灌注法,观测灌注压的变化,可以较客观的掌握酶解消化的程度,利于获取生理状态良好的心肌细胞。且细胞能够进行膜片钳实验。

Isolation of Cardiomyocytes by Flow-Maintained Perfusion for Patch Clamp Studies and Recording of Potassium Channel Currents

Objective To improve the method of isolation of cardiomyocytes in guinea pigs and rats for patch clamp research. Method Cardiomyocytes were prepared by improved flow-maintained Langendorff perfusion method. The currents of potassium channels were recorded by whole-cell patch clamp. Results There were 50% calcium-tolerant cardiomyocytes after exposed to normal calcium concentration solution. A current of potassium channel could be recorded. Conclusion Cardiomyocytes with electrophysiological action can be obtained by the observation of perfusion pressure variation though flow - maintained Langendorff perfusion method.