犬心房肌细胞分离的方法学探讨

Isolation of atrial myocytes from canine

目的建立稳定的适于膜片钳实验研究的犬心房肌细胞分离方法。方法取健康成年犬心脏12个，采用Langendorff灌流，行回旋支插管，正常台式液灌流10s，使心脏自主收缩排除残留余血。持续高钾液灌流，同时结扎其他血管及分支，剪去其余心脏组织，待左房及左心耳充盈，无钙台氏液灌流5min，125U／ml胶原酶Ⅱ200ml反复灌流消化约30～45min，后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，孵育5min后，用200目筛网过滤，逐步复钙法复钙后，室温静置1h，应用于膜片钳记录。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用于膜片钳离子流检测的心房肌细胞。

Objective To establish a practical and reliable technique of atrial myocytes isolation from canine for ion currents research. Method Twelve hearts from healthy canine were intubated through left circumflex branch and 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/m1200 ml for 30 -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 incubated for 5 rain, and then filtered with 200 screen mesh. The isolated myocytes were recovered to normal calcium concentration gradually, and then stored in KB solution at room temperature for 1 h before patch clamp experiments. Result The survival rate of freshly isolated canine atrial myocytes was 90%, which were rod-shaped with clear cross-striations and complete membrane. Conclusion High survival rate and high quality single atrial myocytes can be obtained by our es- tablished skill and are suitable for the t of ion currents by patch-clamp technique.