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目的:寻找一种成年小鼠心肌细胞分离的简便、可行且稳定的方法,以获得耐钙心肌细胞并记录心室肌细胞L型钙电流(IL-Ca)。方法:采用Langerdorff逆行主动脉灌流、无钙台氏液+胶原酶Ⅱ消化法分离成年小鼠单个心室肌细胞;利用含钙细胞外液复钙法筛选耐钙心肌细胞;并采用全细胞膜片钳技术记录心室肌细胞L型钙电流。结果:利用该方法分离心肌细胞可获得90%活细胞;复钙后细胞存活率60%-70%;并可记录到典型L型钙电流。结论:无钙台氏液+胶原酶Ⅱ消化法简单可行且稳定,可分离大量活性较佳且耐钙心肌细胞,可用于心肌细胞离子通道电流记录,从而为心脏电生理研究提供有力的技术支持。
OBJECTIVE: To find a simple, feasible and stable method for the isolation of cardiomyocytes in adult mouse in order to obtain calcium-tolerant cardiomyocytes and record L-type calcium current (IL-Ca) in ventricular myocytes. METHODS: Single ventricular myocytes were isolated from neonatal mouse by Langerdorff’s retrograde aortic perfusion without Ca2 +, collagenase Ⅱ digestion. Calcium-resistant extracellular calcium was used to screen calcium-resistant cardiomyocytes. Whole-cell patch-clamp Technical recording of L-type calcium current in ventricular myocytes. RESULTS: 90% of the viable cells were obtained by this method. The viability of cells after 60% -70% calcium reuptake was recorded. A typical L-type calcium current was recorded. CONCLUSION: Calcium-free Trypsin + collagenase Ⅱ digestion method is simple, feasible and stable, and can isolate a large number of well-activated and calcium-tolerant cardiomyocytes, which can be used for myocardial current channel ion channel recording to provide powerful technical support for cardiac electrophysiological studies .