犬心肌细胞分离方法的改良  

An Improvement for Isolation of Adult Dog Cardiomyocytes

作　　者：刘颖[1] 王雷 彭珍[1] 王洁[1] 王振全 李春梅 宫海滨 

机构地区：[1]徐州市中心医院,江苏徐州221009 [2]徐州市心血管病研究所,江苏徐州221009

出　　处：《现代生物医学进展》2013年第8期1412-1416,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30572073);江苏省生命健康科技专项资金资助(BL2012019);江苏省医学重点人才(RC2007024);徐州市科技发展项目(XZZD1018)

摘　　要：目的:建立高效可靠的成年犬心肌细胞分离方法,获得高产量与高质量的心肌细胞,以便进行犬心肌细胞收缩功能的研究。方法:采用改良的Langendorff灌流胶原酶消化法分离得到左心室心肌细胞。荧光显微镜观察细胞生长状态和形态并利用单细胞收缩动态边缘检测系统测定心肌细胞收缩功能的改变。结果:结果显示,与传统方法相比,即刻分离的心肌细胞状态良好,复钙后的心肌细胞成活率达到70%-80%,转染重组腺病毒β2-EGFP培养48 h,心肌细胞成活率为60%-70%。给予持续电场刺激,心肌细胞可以保持收缩节律和幅度稳定30 min以上。结论:该方法操作简单,分离的活细胞产量高,质量好,节约实验成本和时间,为心血管相关研究提供良好的细胞模型。Objective： To explore an efficient and reliable technique for isolation of adult canine cardiomyocytes. It is necessary to isolate high-yield and high-quality cardiomyocytes in order to observe the contractile function. Methods： Modified Langendorff perfusion method and collagenase digestion method were used in the experiments to obtain cardiomyocytes from left ventricle of canines. Then, their form and structure with fluorescence microscope were observed. The contraction amplitude was measured with video edge tracker method. Results： The results showed, compared with the used method, the state of cardiomyocytes are better in fresh isolation, the survival rate of separated cardiomyocytes was 70%-80% after restoration of normal extracellular Ca2＋ concentration. Transfected by adenovirus β2-EGFP for 48 hours, the survival rate was 60%-70%. The cardiomyocytes were stable of the contraction rhythm and amplitude for 30 min at least after giving sustained electrical stimulation. Conclusion： The improved method isolating adult canine＇s eardiomyocytes is simple and functional which could isolate good quality cardiomycytes. It can save experimental cost and time. It can also offer more significant cell model to the study on the cardiovascular.

关 键 词：心肌细胞 培养 腺病毒 基因 收缩功能 

分 类 号：Q95-3[生物学—动物学] R54[医药卫生—心血管疾病]