大鼠心肌细胞急性分离方法及影响因素探讨

Acute Isolation of Cardiomyocytes in Rat and its Influencing Factors

目的探讨酶解法急性分离大鼠心肌细胞的相关影响因素。方法采用改良Langendorff主动脉逆行灌流法对大鼠全心工作细胞进行酶解分离,分析分离过程中的诸多条件,从中优化最佳的分离方案,为膜片钳实验提供合格细胞。结果分离所用灌流液pH值为7.35~7.40,分离细胞的存活率最高。不同复钙方法对后续细胞存活有明显影响。三步梯度复钙后的细胞存活率明显高于一步、二步复钙法。细胞分离时,灌流速度对细胞存活率有较大影响,3mL/min流速时细胞存活率最高,不同灌流流程对分离细胞的质和量较大影响。采用钙-无钙台氏液先后灌流后再行酶解分离方式可获得长杆状、棱角分明、立体感强的心肌细胞,该细胞复钙后存活率为(56.8±2.6)%;直接使用无钙台氏液灌流后再行酶解分离方法获得(57.2±3.3)%的心肌细胞,其复钙后存活率为(36.7±3.5)%,前述方法明显优于后者。结论灌流液的pH值、灌流速度、细胞分离后的复钙方法及不同的灌流流程等因素可对大鼠心肌细胞的急性分离结果产生显著影响。

Objective To explore the influencing factors of acute isolation of cardiomyocytes in rat by enzymatic hydrolysis.Methods The modified Langendorff aortic retrograde perfusion method was used to isolate the whole-hearted working cells in rats.The best separation conditions were optimize by exploring them one by one,and qualified cells were provided for patch clamp experiment.Resluts The survival rate of isolated cells was the highest when the pH ranged from7.35 to 7.40.In addition,the different methods of recalcification had the obvious effects on the survival of the subsequent cells.Among them,the survival rate of the cells with the three step gradient of recalcification was significantly higher than others.Meanwhile,the cell separation of perfusion speed had also a great influence on the cell survival rate.The experiment showed that the survival rate of the cells was the highest at a flow rate of 3 mL/min.In addition,different perfusion processes had a greater effect on the quality and quantity of the isolated cells.After enzymolysis separation followed by the perfusion of Ca^（2＋）-Ca^（2＋） free Tyrode＇s solution,the long rod-shaped,clear-cornered,and highly stereotypic cardiomyocytes were obtained.The survival rate of cardiomyocytes was（56.8±2.6）% after recalcification.After enzymolysis separation followed by the perfusion of calcium-free Tyrode＇s solution,（57.2±3.3）% of cardiomyocytes were obtained and the survival rate was（36.7±3.5）% after recalcification.The former method was obviously better than the latter.Conclusion The perfusate pH,perfusion rate,the method of recalcification after cell separation,and different perfusion processes had a significant impact on the acute isolation of cardiomyocytes in rat.