用于钙信号研究的小鼠心室肌细胞急性分离的优化  

Study on an optimized method for acute isolation in mice ventricular myocytes for calcium signal

作　　者：管倩楠 段斐[1] 吴银玲[1] 王玉娟[1] 张雪娇[1] 

机构地区：[1]河北大学医学院,河北保定071000

出　　处：《医学研究与教育》2016年第2期6-11,共6页Medical Research and Education

基　　金：河北省医学科学研究课题计划项目(20130049)

摘　　要：目的建立一种可快速获取生理状态良好的用于钙信号研究的小鼠心室肌细胞的方法,同时比较两种细胞保存液对心室肌细胞钙信号的影响。方法取成年小鼠心脏,用改进的Langendorff心脏灌流系统行主动脉逆行灌流,利用酶联合法消化,取得心室肌细胞置于两种不同的细胞保存液中,在显微镜下观察并检测其状态。采用共聚焦显微镜观测细胞内钙信号,记录钙火花。结果该方法可以在较短的时间内得到65%以上的长杆状心室肌细胞,细胞能够正常存活12~24 h,并且可以立即用于钙火花观测实验研究。比较两种保存液,细胞保存液A中富含高钾离子,细胞保存液B中含有正常的胞外钙离子浓度,观察到的钙火花细胞保存液B中比A液的频率高、幅度大,在达峰时间上比A液的时间短。结论通过严格控制各项实验条件,能够成功得到存活率高、纯度高、耐钙的可用于钙火花观测的小鼠心室肌细胞。细胞保存液中的钾离子含量少,并有适量的钙离子有助于观测钙火花的发生。Objective To introduce an optimized method for acute isolation in mice ventricular myocytes for signal research,meanwhile compare the calcium spark differences in two kinds of cell preservation medium. Methods Hearts were excised quickly from anesthetized adult mice, then aortic was perfused in counter-current with the optimized heart Langendorff apparatus. Digestive enzyme combined method was applied to obtain the ventricular myocyte in two different types of cell preservation solution, intracellular calcium signals were observed with confo- cal microscope and the calcium sparks were recorded. Results This method could get more than 65 % long rod of myocardial cells in a relatively short period of time surviving more than 12-24 h normally,and could be used im- mediately to observe calcium sparks experiments. The difference between cell preservation medium A and B was that A had a higher concentration of K^＋ and B had the same Ca^2＋ concentration as the extracellular fluid did. There were higher frequence, amplitude and shorter time-to-peak, full duration at half maximum of Ca^2 ＋spaker in cell preservation medium B than those in cell preservation medium A. Conclusion Through strictly controlling variously experimental conditions, the myocardial cells of mice can be successfully obtained with higher survival rates,purity and calcium-tolerant,which would be convenient to observe the calcium sparks. It is helpful to ob- serve the occurrence of calcium sparks with less potassium and moderate calcium in cell preservation medium.

关 键 词：心室肌细胞 分离 钙火花 心血管疾病 

分 类 号：R331[医药卫生—人体生理学]