成年小鼠心肌细胞分离技术  

Isolation of cardiomyocytes from the adult mouse heart

作　　者：张然[1] 余志斌[1] 王云英[1] 

机构地区：[1]第四军医大学航空航天生理学教研室,西安710032

出　　处：《生理学报》2004年第5期656-660,共5页Acta Physiologica Sinica

基　　金：This work was supported by the National Natural Science Foundation of China (No. 30370538).

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.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 I at 37 oC and the enzymatic digestion was terminated immediately when the perfusion pressure was lowered to 28 mmHg. The heart was then cut off the cannula and the atria and aorta dissected away. The ventricular tissue was chopped and the single myocyte was dispersed gently by a wide tipped pipette. The viability of freshly isolated cardiomyocytes was more than 70 %. The cardiomyocytes were kept in Joklik’s minimum essential medium containing 1 % BSA and 10 mmol/L BDM, then extracellular calcium was restored step-wise to a final concentration of 1.25 mmol/L. The viability of cardiomyocytes reduced to (40~50) % after 4 h standing. More than 90 % of rod-shaped cardiomyocytes were quiescent and had visible cross striations and sharp edges. The amplitude of unloaded shortening in cardiomyocytes was (9.72±0.43) % during 1.0 Hz stimulation, (11.28±0.43) % at 2.0 Hz and (11.40±0.45) % at 5.0 Hz. These results indicate that high yield and high quality cardiomyocytes can be obtained. In addition, the standards of identifying cardiomyocyte quality are concise and are suitable to culture the cardiomyocytes or to study the physiological function of cardiomyocytes.

关 键 词：小鼠 心肌细胞 分离 

分 类 号：Q952[生物学—动物学]