原代乳鼠心肌细胞分离培养的改进

包馨慧, 李晓梅, 杨毅宁, 马依彤, 陈邦党, 陶静, 陈小翠

(新疆医科大学第一附属医院心脏中心, 乌鲁木齐830054)

摘要:目的探讨提高细胞纯度、存活率及活性的乳鼠心肌分离培养方法。方法改进心脏组织的处理方法，用胰酶预处理后再用II型胶原酶依次处理心肌组织，采用差速贴壁及化学药物纯化心肌细胞。结果提取的单个心肌细胞结构完整，呈类圆形；12 h时大部分细胞贴壁；24 h后几乎全部贴壁，仅见少量漂浮细胞，贴壁细胞伸出伪足，呈梭形、三角形等，细胞出现自主搏动。48 h后心肌细胞伪足间形成联接，交织成网状，局部心肌细胞出现同步搏动。3 d后心肌细胞聚集成簇，呈岛屿样搏动。心肌细胞存活率为96.6%，纯度>96%。结论此改进方法所提取培养的乳鼠心肌细胞存活率及纯度高，细胞活性好，结构功能完整，可用于相关实验中原代乳鼠心肌细胞的分离培养。

关键词:心肌细胞； 原代培养； 新生大鼠

中图分类号：Q463文献标识码：A

doi:10.3969/j.issn.1009-5551.2015.05.010

[收稿日期：2014-07-02]

基金项目：国家药典委员会国家药品(维药)标准提高项目(510)

作者简介：刘杰(1980-)，男，硕士，主管中药师，研究方向：中药质量控制。

基金项目：国家自然科学基金(81260624)

作者简介：木克热木·吐地买提(1988-)，女(维吾尔族)，在读硕士，研究方向：食品毒理学。

An improved protocol for primary cultur of myocytes extracted from neonatal rat

BAO Xinhui, LI Xiaomei, YANG Yining, MA Yitong, CHEN Bangdang, TAO Jing, CHEN Xiaocui

(DepartmentofCardiology,TheFirstAffiliatedHospitalofXinjiangMedicalUniversity,

Urumqi830054,China)

Abstract:ObjectiveTo improve the traditional method of culturing neonatal rat myocytes for the better purity, survival rate and activity of myocytes in experiments. MethodsTrypsin and type Ⅱ collagen enzyme were used to digest heart tissue and myocytes were purified with differential sticking wall method and chemical purification. ResultsThe extracted single myocyte is of structural integrity and circular shape. Most cells were touching to wall in 12 hours, and all cells sticking to wall in 24 hours, with small amount of floating cells. The touching-wall cells extended pseudopodia in the form of fusiformis, triangle and so on, with independent rhythm. Myocardial cells′ pseudopodia became connected into webs 48 hours later, with local synchronization rhythm. There days later, myocytes gethered into clusters, with island-like pulse. The activity and purity of myocytes was up to 96%. ConclusionThis method helped improve the activity and purity of myocytes, and protect the integrity of cells. The extracted myocytes could be used in relative experiments.

Key words: myocardial cell; primary culture; neonatal rat

乳鼠心肌细胞常被用作体外实验研究模型，用于心血管病的基础研究。虽然早在1960年，Harary等[1]已提出原代乳鼠心肌细胞分离培养方法，但由于这一传统方法所分离的心肌细胞存活率及纯度低、细胞活性差等原因，尚不能满足于基础研究的需求。目……