论文部分内容阅读
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 L的20 mmol.L 1CaCl2,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 investigate the acute isolation of cardiomyocytes from Kunming mice and the recording of action potential and L-type calcium channel current. Methods Three-step perfusion method was used. Firstly, calcium-free Tyrode’s solution was perfused and then replaced with calcium-free table containing type Ⅱ collagenase 0.1 mg.mL 1, trypsin 0.01 mg.mL 1 and bovine serum albumin 0.2 mg.mL 1 During the perfusion of liquid and digestive fluid, 20 mmol·L -1 CaCl 2 20 L was added every 5 min to observe whether there was a single cardiomyocyte in the effluent to determine the end point of digestion. Finally, the perfusate containing 1 mg.mL -1 bovine serum albumin KB solution, using whole-cell patch clamp recording of action potential and L-type calcium channel current. RESULTS: 80% -90% of rod-shaped cardiomyocytes were obtained. After rehydration, 60% of the cells remained still, the surface of the cells remained clean and tidy, the refraction was strong, the edges and stripes were clear, Ventricular myocytes, and recorded a typical action potential, L-type calcium channel current. Conclusion The cells isolated by this separation method have calcium tolerance and normal electrophysiological properties.