成年小鼠心肌细胞分离技术-生理学报.PDFVIP

656 生理学报 Acta Physiologica Sinica , October 25, 2004, 56 (5): 656-660 生理学报 Acta Physiologica Sinica , October 25, 2004, 56 (5): 656-660 实验技术 成年小鼠心肌细胞分离技术 * 张 然，余志斌 ，王云英 第四军医大学航空航天生理学教研室，西安 710032 摘 要: 为进行成年小鼠心肌细胞培养与收缩功能研究, 首先必须获得高产量与高质量的心肌细胞。本实验采用Langendorff 装置行恒流灌流心脏, 同时监测灌流压力的变化。根据小鼠鼠龄微调灌流流速, 使初始灌流压力保持在40 mmHg 。用0.05 % 单 一粗制胶原酶在 37 ℃条件下消化心脏, 当灌流压力下降至28 mmHg 时, 即刻终止消化。轻轻吹散心肌细胞后, 用含 1 % 牛血 清白蛋白的 Joklik’s MEM 培养液保存，逐步法恢复细胞外钙离子浓度。获得的心肌细胞存活率大于 70 ％，复钙后耐钙 心肌细胞静置 4 h ，心肌细胞存活率仍能保持在(40~50) % 。其中，90 ％以上存活的长杆状心肌细胞无明显搏动，细胞膜 表面光滑，横纹清晰，两端边缘锐利, 折光性较强, 复钙后保存4 h 或 5.0 Hz 刺激 5 min 后，仍能保持正常形态。在 1.0 Hz 刺激条件下, 心肌细胞缩短幅度为(9.72 ±0.43) %; 2.0 Hz 刺激下为(11.28 ±0.43) %; 在 5.0 Hz 刺激下, 达到(11.40 ±0.45)% 。 这些结果表明, 采用本方法可获得高产量与高质量的成年小鼠心肌细胞, 且易于操作, 重复性较好。 关键词: 小鼠; 心肌细胞; 分离 中图分类号: Q463 Isolation of cardiomyocytes from the adult mouse heart * Zhang Ran, YU Zhi-Bin , WANG Yun-Ying Department of Aerospace Physiology, Fourth Military Medical University, Xi’an 710032, China Abstract: In order to culture cardiomyocytes or to observe the contractile function of adult mouse cardiomyocytes, it is necessary to isolate high-yield and high-quality cardiomyocytes at first. The mouse was injected with heparin (5 000 IU/kg, i.p.) 20 min prior to the experimental protocol, then was sacrificed by cervical dislocation. The heart was excised and the aorta was cannulated rapidly. The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and perfusion pressure was monitored. The initial perfusion pressure was maintained at 40 mmHg by regulating the flow rate. The heart was digested by 0.05 % crude collagenase