上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (4): 520-.doi: 10.3969/j.issn.1674-8115.2011.04.033

• 技术与方法 • 上一篇    下一篇

新生大鼠心肌细胞分离、纯化及培养方法的改进

庞勇军1,2, 孙 莉3, 谢文娟4, 莫书荣2   

  1. 1.桂林医学院生理学教研室, 桂林 541004； 2.广西医科大学生理学教研室, 南宁 530021；3.桂林医学院组织学与胚胎学教研室, 桂林 541004； 4.桂林医学院生药学教研室, 桂林 541004
  • 出版日期:2011-04-28 发布日期:2011-04-28
  • 通讯作者: 莫书荣, 电子信箱: mosr@21cn.com。
  • 作者简介:庞勇军（1972—）, 男, 讲师, 硕士；电子信箱: pangyongjungl@163.com。
  • 基金资助:

    广西自然科学基金（0728140）

Improvement of method for separating and culturing neonatal rat ventricular myocytes

PANG Yong-jun1,2, SUN Li3, XIE Wen-juan4, MO Shu-rong2   

  1. 1.Department of Physiology, Guilin Medical University, Guilin 541004, China；2.Department of Physiology, Guangxi Medical University, Nanning 530021, China；3.Department of Histology and Embryology, 4.Department of Pharmacognosy, Guilin 541004, China
  • Online:2011-04-28 Published:2011-04-28
  • Supported by:

    National Science Foundation of Guangxi, 0728140

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摘要：

目的 应用酶消化法消化心室肌组织，培养心肌细胞，探索获得高存活率和高纯度心肌细胞的培养条件，为生理学和药理学研究提供细胞模型。方法 胰蛋白酶和胶原酶Ⅰ联合消化新生大鼠心室肌组织，控制消化时间和消化温度；用差速贴壁法和化学抑制法纯化心肌细胞进行体外培养。荧光显微镜观察心肌细胞形态学变化；流式细胞仪检测肌钙蛋白Ⅰ的表达；Fluo-3/AM进行细胞质内游离钙离子染色。结果 培养的心肌细胞4 h后开始贴壁生长，24 h后有少数单个细胞出现自发性节律性搏动，之后连接成片，细胞簇同步收缩，细胞形态、结构完整；肌钙蛋白Ⅰ阳性率为91%；所有心肌细胞细胞质内钙离子染色阳性，荧光强度随细胞搏动呈强弱周期性变化。结论 应用酶消化法可简单快捷地获得大量活力好、纯度高的心肌细胞，为心血管相关研究提供良好的实验模型。

关键词: 心肌细胞, 分离, 原代培养, 胶原酶, 大鼠

Abstract:

Objective To culture neonatal rat ventricular myocytes after enzyme digestion, explore method to obtain myocardial cells with high survival rate and high purity, and offer cell model for physiological and pharmacological study. Methods Neonatal rat ventricular tissues were digested by trypsinase and collagenaseⅠ, the digestion time and temperature were controlled. Myocardial cells were purified by adherence separation method and chemical inhibition method, and were cultured in vitro. Morphocytology changes were observed by fluorescence microscopy, troponinⅠ was determined by flow cytometry, and free Ca2+  in cytoplasm of cardiomyocytes was detected with Fluo-3/AM. Results Cardiomyocytes began to adhere and grow after 4 h, few individual cells began to beat spontaneously after 24 h, and cardiomyocytes got together and beat synchronously in clusters gradually, with the cellular shape and structure being intact. The positive rate of troponinⅠ was 91%, Ca2+ staning was positive in cytoplasm of all cardiomyocytes, and periodic variation was detected along with cell pulsation. Conclusion A large number of vigorous cardiomyocytes with high purity can be obtained by enzyme digestion method, which may provide favourable model for cardiovascular research.

Key words: cardiomyocyte, isolation, primary culture, collagenase, rat