摘要
目的探讨乳鼠心肌细胞的原代培养方法,更好地获取高存活率、高搏动率及高纯度的心肌细胞,以建立良好的原代心肌细胞研究模型。方法无菌状态下取出生3 d以内Wistar乳鼠心室肌,以混合消化酶(0.06%胰蛋白酶和0.1%Ⅱ型胶原酶均量混合液)反复进行短时间多次消化,用差速贴壁法纯化心肌细胞,并加入5-溴脱氧尿嘧啶抑制非心肌细胞分裂增殖,2 d后首次换液,以后隔日换液。结果心肌细胞24 h左右已基本贴壁,并可见单个细胞搏动,2～6 d形成单层心肌细胞和其细胞簇的同步搏动,经培养所获细胞搏动良好,存活率高,免疫组化鉴定显示培养的心肌细胞纯度95%以上。结论该方法是一种较为理想的乳鼠原代心肌细胞培养方法,值得进一步推广。
Objective To explore the methods of the primary culture of cardiocytes for the sucking mice so as to better obtain the high livability,high pulsating rate and high purity of the cardiocytes and establish the positive study model of primary-cultured cardiocytes.Methods Under the germ-free condition,the ventricular muscle was collected from the 3 d-born Wistar sucking mice.The mixed digestive enzyme solution（the mixture of trypsin 0.06% and II-type collagenase 0.1%,equal dose）was used for the repeated digestion in a short time.The differential adhesion was adopted to purify the cardiocytes.5-bromodeoxyuridine was used to inhibit the division and proliferation of non-cardiocytes.In 2 days,the solution was replaced for the first time and it was replaced once every two days since then.Results In about 24 h,the cardiocytes were adherent basically.The single cell was pulsated visibly.In 2 to 6 d,the monolayer cardiocytes were formed and the cell cluster was pulsated synchronously.Through culturing,the cells obtained were pulsated well.The livability was high.The immunohistochemical identification showed that the purity of the cultured cardiocytes was over 95%.Conclusion This method is satisfactory for the primary culture of the cardiocytes for the sucking mice and it deserves to be promoted.
出处
《世界中西医结合杂志》
2012年第4期293-294,319,共3页
World Journal of Integrated Traditional and Western Medicine
基金
国家自然基金资助项目(No.81072965)
关键词
心肌细胞
原代培养
乳鼠
Cardiocytes
Primary Culture
Sucking Mice
