昆明小鼠心室肌细胞分离方法及动作电位、L型钙通道电流记录  

Isolation of Calcium-tolerant Cardiomyocytes from KM Mouse and Recording of Action Potential and the Currents of L-type Calcium Channels

作　　者：李映新[1] 黄媛恒[2] 林兴[3] 黄仁彬[1] 

机构地区：[1]广西医科大学药学院,南宁530021 [2]广西医科大学基础医学院实验生理学科学实验中心,南宁530021 [3]广西医科大学医学科学实验中心,南宁530021

出　　处：《中国现代应用药学》2013年第6期581-586,共6页Chinese Journal of Modern Applied Pharmacy

基　　金：广西科学研究与技术开发计划项目(桂科攻0630002-2A);广西中医药科技专项课题(GZKZ10-122);2011年广西研究生创新计划项目(2011105981002D26)

摘　　要：目的 探讨耐钙昆明小鼠心室肌细胞的急性分离方法及动作电位、L型钙通道电流的记录。方法 采用三步灌流法，首先灌流无钙台氏液，再换成含Ⅱ型胶原酶0.1 mg·mL^-1、胰蛋白酶0.01 mg·mL^-1、牛血清白蛋白0.2 mg·mL^-1的无钙台氏液灌流，消化液灌流期间，每隔5 min加入20 mL 的20 mmol·L^-1 CaCl2，以观察流出液是否有单个心肌细胞来判断消化终点，最后灌流含1 mg·mL^-1牛血清白蛋白的KB液，采用全细胞膜片钳记录方式记录动作电位及L型钙通道电流。结果 获得80%-90%杆状心肌细胞，复钙后，仍有60%细胞保持静止，细胞表面干净整洁，折光性强，边缘和横纹清晰，立体感强，获得60%左右的耐钙心室肌细胞，并记录到典型的动作电位、L型钙通道电流。结论 该分离方法分离的细胞具有耐钙性和正常电生理特性。OBJECTIVE To explore and establish simple and reliable method of isolating single calcium-tolerant ventricular myocytes of KM mouse for patch clamping and recording of action potential and the currents of L-type calcium channels. METHODS The three-step enzymatic dissociation method was used to isolate myocytes from ventricular tissue by the Langendorff apparatus. Hearts were perfused retrogradely with Ca2＋ free Tyrode’s solution initially, and then with Ca2＋ free Tyrode’s solution contained 0.1 mg·mL^-1 collagenase Ⅱ, 0.01 mg·mL^-1 trypsin and 0.2 mg·mL^-1 bovine serum albumin. During the perfusions, 20 mL 20 mmol·L-1 of CaCl2 was added to the digestive juice every 5 minutes. The terminal of digestion was judged by observing the existence of single myocyte in the efflux solution. KB solution contained 1 mg·mL^-1 bovine serum albumin was ultimately used. The action potential and the currents of L-type calcium channels were recorded by patch clamp in the entire cell mode. RESULTS The 80%-90% cells obtained were rod-shaped myocytes. After the recalcification, 60% cells stayed still, and the action potentials and L-type calcium channel currents could be successfully recorded. CONCLUSION The method is economical and effective, the myocytes obstaied in this way are suitable for the recording of patch clamp technique.

关 键 词：昆明小鼠 心肌细胞 膜片钳 动作电位 L型钙电流 

分 类 号：R331.3[医药卫生—人体生理学]