动脉平滑肌细胞和心肌细胞钾通道的膜片钳全细胞记录技术  

A technique of whole-cell recording for studying potassium channels in isolated cells from artery and ventricle of heart

作　　者：崔文玉[1] 陈玉萍[1] 汪海[1] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850

出　　处：《军事医学科学院院刊》2003年第6期452-456,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家"十五"重大科技专项课题 ( 2 0 0 2AA2Z3 13 7)

摘　　要：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 simple method of acutely preparing isolated single ventricular myocytes and arterial smooth muscle cells and then to study the potassium channels with whole-cell configuration patch clamp technique in these cells. Methods:Single isolated smooth muscle cells from rat intrapulmonary artery were obtained by employing collagenaseⅠ(1?mg/ml) and papain (1?mg/ml). Single rat tail arterial smooth muscle cells were prepared by enzymatic digestion with pronase E (1?mg/ml) and collagenaseⅠ(1?mg/ml).The ventricular myocytes were prepared from guinea pig by the enzymatic dissociation with pronase E (0.5?mg/ml) solutions.Results:The isolating method could produce adequate cells in normal form. When stored at 4℃, the dissociated cells held their responsiveness for 8h after isolation. The cells with smooth borders were chosen for whole-cell recording. The evoked outward currents could be fully depressed by potassium channel blockers CsCl and TEA.Conclusion:The enzymatic dissociation method is a very good technique which is easy to handle and needs simple instruments and low expense for patch clamp research.

关 键 词：肌 平滑 血管 动脉平滑肌细胞 心室肌细胞 急性酶分离 膜片钳术 钾通道 

分 类 号：R331[医药卫生—人体生理学]