适用于膜片钳实验的犬心房肌细胞的分离方法分析研究

Isolation of atrial cardiomyocytes from adult canine for Patch-clamp studies

目的建立稳定的成年犬心房肌细胞的分离方法,进行膜片钳实验的研究。方法采用Langendorff灌流,台氏液灌流10s后无钙台氏液灌流5min,125U/ml胶原酶Ⅱ反复灌流消化约25～45min,用无钙台氏液冲洗心脏5min,剪下心房肌组织,KB液中室温下剪碎,吹打,温育5min后,用200μm筛网过滤,室温静置,逐步复钙法复钙后,进行膜片钳实验。结果分离的活性心肌细胞比率约90%,形态呈杆状、横纹清晰、膜周边光滑完整。结论采用本方法可以获得高产量与高质量的适于膜片钳实验的成年犬心房肌细胞。

Objective To establish a practical and reliable technique of isolation atrial cardiomyocytes from adult canine for patch clamp research.Methods Hearts were perfused for 10 s by the Langendorff perfusion apparatus with normal Tyrode’s solution,then with Ca2＋-free Tyrode’s solution for 5 min and with enzyme solution containing type Ⅱ collagenase 125 U/ml for 25~45 min,atrium were washed by Ca2＋-free Tyrode’s solution for 5 min,after which the atrium were minced into small pieces in KB solution,dispersed and filtered.The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results The survival rate of freshly isolated adult canine atrial and ventricular myocytes were 90%,rod-shaped with clear cross-striations and coplete membrane.Conclusion High yield and high quality single atrial cardiomyocytes could be obtained ,and were suitable for patch-clamp technique.