犬心房肌细胞分离方法的改良及影响因素探讨

Research on Improvement of Method Isolating Canine Atrial Cardiomyocytes

目的建立高效稳定的犬心房肌细胞分离方法,获得高质量的心房肌细胞,为研究离子通道疾病提供合格的单个细胞。方法健康成年犬12只,一种方法在心脏左回旋支处插管,剪去左右心室以及室间隔组织,含钙台式液灌流(改良方法),另一种方法在心脏左回旋支处插管后固定,无钙台式液灌流,再剪去多余组织(传统方法),灌注胶原酶分离心房心肌细胞。倒置相差显微镜下观察心肌细胞形态变化,台盼蓝染色法检测细胞存活率,膜片钳技术检测起搏电流。结果两种方法比较,改良方法心房肌细胞的成活率显著高于传统方法(P<0.05),细胞的自主收缩率显著低于传统方法(P<0.05);胶原酶的消化时间显著减少(P<0.05),并且胶原酶的使用量少(P<0.05)。改良方法分离的心房肌细胞形态呈杆状,边缘锐利,横纹清晰状态良好,并可以记录到完整的起搏电流。结论改良方法操作简单,分离心房肌细胞成活率高,可以记录到稳定、完整的起搏电流,节约实验成本和时间,为心房离子通道疾病相关研究提供良好的细胞模型。

Objective To explore an efficient and reliable method for isolating high-quality single cardiomyocytes from atrium of canine for research on ion channel diseases.Methods Twelve hearts of healthy adult canine were used in comparing the two isolation methods.In‘traditional method’,Langendorff perfusion was performed after intubation at the cyclotron branch and fixation.Then,the excess tissues of the heart were cut off and the left atrial myocytes were isolated through collagenase digestion.Regarding the‘improved method’,after intubation at the cyclotron branch,right ventricle and interventricular septal tissue were cut off followed by Langendorff perfusion.The morphology of cardiomyocytes was observed by optical microscopy and the cardiomyocyte viability was evaluated by trypan blue.Pacing current was measured by patch clamp technique.Results Compared to traditional method,the survival rate,autonomic shrinkage rate and cell morphology were significantly improved(P<0.05 respectively).The cardiomyocytes isolated using improved method tended to be rod-shaped with clear cross-striations and complete membrane.Complete pacing current could be recorded using patch-clamp.Conclusion Improved method for isolating cardiomyocytes is simple and time and cost saving,and provided a good cell model for the research on atrial ion channel diseases.