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.
出处
《中国现代应用药学》
CAS
CSCD
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)
关键词
昆明小鼠
心肌细胞
膜片钳
动作电位
L型钙电流
KM mouse
ventricular myocyte
patch clamp technique
action potentials
L-type calcium channel currents