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目的 :介绍一种动脉平滑肌、心室肌细胞急性酶分离方法 ,并在该分离技术的基础上采用膜片钳全细胞记录方式研究细胞钾离子通道的特性。方法 :采用胶原酶Ⅰ (1mg ml)、木瓜蛋白酶 (5mg ml)消化分离大鼠肺内动脉平滑肌细胞 ;用链霉蛋白酶E(1mg ml)、胶原酶Ⅰ (1mg ml)消化分离大鼠尾动脉平滑肌细胞 ;用链霉蛋白酶E(0 .5mg ml)灌流消化、分离得到豚鼠心室肌细胞。结果 :以上分离的细胞数量多 ,形态正常 ,胞壁光滑完整 ,活性好。于4℃无钙液中保存 ,8h内可用于膜片钳全细胞记录。记录出的外向电流可被钾通道阻断剂CsCl及TEA所阻断。结论 :用酶急性分离的动脉平滑肌和心室肌细胞应用于膜片钳研究中具有快速、经济、简便易行等优点。
OBJECTIVE: To introduce a method for the acute enzyme separation of arterial smooth muscle and ventricular myocytes, and to study the characteristics of potassium channel by patch-clamp whole cell recording based on the separation technique. Methods: Pulmonary artery smooth muscle cells of rats were digested with collagenase Ⅰ (1 mg ml) and papain (5 mg ml), and the tail artery of rat was digested with streptavidin E (1 mg ml) and collagenase Ⅰ (1 mg ml) Smooth muscle cells were isolated and perfused with Pronase E (0.5 mg ml), and isolated from guinea pig ventricular myocytes. Results: The number of cells isolated above, normal morphology, cell wall smooth and complete, good activity. Calcium-free solution stored at 4 ℃, 8h can be used for patch-clamp whole cell recording. The outward currents recorded can be blocked by potassium channel blockers CsCl and TEA. Conclusion: It is rapid, economical, simple and easy to use in the study of patch clamp in acute arterial smooth muscle and ventricular myocytes isolated by enzyme.