原代乳鼠心肌细胞培养方法的改进  

An improved protocol for primary culture of cardiomyocyte from neonatal rat

作　　者：陶静[1] 马依彤[1] 李晓梅[1] 杨毅宁[1] 马翔[1] 陈邦党 刘芬 陈小翠 盖敏涛 

机构地区：[1]新疆医科大学第一附属医院心脏中心,乌鲁木齐830054 [2]新疆心血管病研究所重点实验室

出　　处：《中华心血管病杂志》2014年第1期53-56,共4页Chinese Journal of Cardiology

基　　金：国家自然科学基金一地区科学基金项目（81060025,81060021）;教育部高等学校博士学科专项科研基金项目（20106517110002）;973计划前期研究专项（2010CB535013）;新疆研究生科研创新项目（XJGR2013086）

摘　　要：目的通过对传统乳鼠心肌细胞体外培养的条件、方法进行改进，得到纯度高、活力好、结构功能完整的心肌细胞。方法改进心室肌组织剪切方法，分别用胰酶、Ⅱ型胶原酶依次消化分离心肌组织，严格要求所需溶液pH值，差速贴壁法和化学抑制法相结合纯化心肌细胞。结果心肌细胞12h后开始贴壁生长，24h出现单个细胞自发性搏动，48h部分心肌细胞间形成交联，72h心肌细胞黏合成簇，96h后细胞伪足相互接触交织成密集的网，形成放射状排列的细胞簇，可见岛屿样搏动。细胞存活率、纯度均达98％以上。结论此种方法培养的心肌细胞纯度高、活力好、结构及功能完整，是一种可靠的原代乳鼠心肌细胞培养方法。Objective The present study describes an improved in vitro cuhure method for obtaining high purity, vital and fully functional cardiomyocytes from neonatal rat. Methods After cutting ventricular tissue with improved method, ventricular tissues were digested with low concentrations of trypsin overnight at 4 °C, and then underwent collagenase II digestion. Thereafter, cardiomyocytes were purified by combined differential adhesion and chemical inhibition methods. Results Adherent cardiomyocytes were seen at 12 h after culture, spontaneously beating cardiomyocytes were observed at 24 h after culture, crosslinked cardiomyocytes were found at 48 h after culture, adhesion clustered cardiomyocytes were seen at 72 h after culture, dense network formed from inter-connected was evidenced together with radial arranged cell clusters and cell pseudopodia 96 h the mutual contact woven into and formed radically ordering cell clusters and island-like beating cardiomyocytes at 96 h after culture. The cell survival rate and purity were more than 98%. Conclusion Fully functional spontaneous beating cardiomyocytes can be obtained by the use of this improved primary neonatal rat cardiomyocytes culture method.

关 键 词：肌细胞 心脏 细胞培养技术 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]