摘要
目的对传统的原代乳鼠心肌细胞分离方法进行改良,获得活性好、纯度高、反应性好的原代乳鼠心肌细胞。方法利用断头开胸取心法取新生24 h内的Wistar大鼠心肌组织,无菌剪碎后用0.25%不含EDTA胰酶混合II型胶原酶消化法,采用自制消化组合装置分离消化细胞。逐日在倒置相差显微镜下观察细胞状态。采用心肌细胞特异性蛋白α-横纹肌肌动蛋白(α-actin)单克隆抗体对培养的心肌细胞进行免疫荧光鉴定。另外使用异丙肾上腺素和去甲肾上腺素作用于细胞,观察细胞反应情况。结果培养后24 h即可观察到部分细胞搏动,可初步确定为心肌细胞;48 h后细胞逐渐展开,连续观察20 d,发现心肌细胞在3~14 d期间搏动频率与形态无明显变化。对比观察发现断头开胸取心法获取的原代心肌细胞中红细胞数量明显减少。免疫荧光鉴定发现,本方法获得的心肌细胞纯度高达95%。此外,分离的原代乳鼠心肌细胞对异丙肾上腺素和去甲肾上腺素反应灵敏。结论断头开胸取心法配以混合酶液消化心脏组织分得的原代乳鼠心肌细胞反应性好、纯度高、有活力,可满足大多数实验的要求。
Objective To improve the traditional isolation method of primary cardiomyocytes in neonatal rats to get neonatal rat cardiomyocytes with good activity,high purity and good reactivity. Methods Neonatal Wistar rats myocardial tissue within 24 h were obtained with broken head-open chest method,then digested using0. 25% trypsin without EDTA mixing collagenase II,as well as using temperature magnetic stirrer and a rotor in tissue suspension during digestion process. Cell shape observed under inverted phase contrast microscope every day. Using cardiac cell-specific protein α-Sarcomeric actin( α-actin) monoclonal antibody,the cultured myocardial cells were identified with immunofluorescence. Primary neonatal rat cardiomyocytes were treated with clinical common drugs,isoprenaline and noradrenalin,then cell responses were observed. Results Myocardial cells were confirmed for some cells beating after 24 h culturation and gradually expanded after 48 h,through 20 d continuous observation,no significant changes of cardiomyocytes beat frequencies and shapes were found from 3 rd to 14 th day. The number of red blood cells in the primary cardiac cells obtained with the method of the broken head-open chest was significantly reduced. Immunofluorescence identification showed the purity of primary neonatal rat cardiomyocytes achieved with new method was up to 95%. In addition,isoprenaline and noradrenalin,could make cell response sensitively. Conclusion Primary neonatal rat myocardial cells isolation with the broken head-open chest method are more sensitive and active,and of higher purity,it can meet the requirements of most experiments.
出处
《航天医学与医学工程》
CAS
CSCD
北大核心
2016年第3期163-168,共6页
Space Medicine & Medical Engineering
基金
国家自然科学基金(31401006)
"973"计划前期研究专项(2014CB560704)
山西医科大学博士启动基金(03201407)
关键词
乳鼠
断头开胸取心
原代心肌细胞
混合酶液消化
鉴定
活性
neonatal rat
broken head-open chest
primary cardiomyocytes
hybrid enzyme digestion
identification
activity