多用途成年大鼠心室肌细胞分离方法

目的 建立稳定的可同时用于细胞培养和膜片钳实验的成年大鼠心室肌细胞的分离方法 .方法 应用Langendorff灌流.生物酶消化法分离耐钙心肌细胞.分别用左右心室肌做细胞培养和全细胞膜片钳研究.结果 左室心肌细胞数(3.7±0.6)× 10~6,杆状细胞得率(84.85±2.7)%.右室心肌细胞横纹清晰,折光率好,膜片钳实验时容易封接破膜,记录到典型的I_(Ca).L、I_(K1)、I_(to)、I_(Na)电流.结论 该方法 简单、节约、重复性好,改善了杆状细胞得率、细胞质量,左右心室肌细胞分别能很好地用于细胞培养和膜片钳实验.

Isolation of Adult Rat Ventricular Myocytes for Culture and Experiment of Patch Clamp

Objective To improve current enzymatic methods to isolate a high yield of high-quality adult rat ventricular myocytes for both culture and experiment of patch clamp.Methods Calcium tolerant ventricular myocytes were isolated with collagenase Ⅱ by Langendorff perfusion.Left and right ventricular myocytes were used respectively for culture with or without FBS and patch clamp.The currents of L-type calcium channels were recorded by patch clamp in the entire cell mode.Results By using this method,we routinely obtained a high yield (3.7±0.6)× 10~6/left ventricle] and high percentage(84.8±2.7)%of rod-shaped myocytes.most of which were clearly defined sarcomeric striations and quiescent state.A typical current of I_(Ca) L,I_(K1),I_(to) and I_(Na) was recorded in the myocytes from right ventricle.Conclusion This is a simple and reliable myocyte isolation method that greatly improves the yield,cell quality,and reproducibility of ventricular myocytes isolation,and is suitable to both culture and patch clamp.