中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (20): 3683-3687.doi: 10.3969/j.issn.1673-8225.2011.20.018

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

C57胎鼠、乳鼠及成年小鼠心室肌细胞分离、培养及鉴定

张  玲1，段明军2，魏  琴2，陈  华1，时  利3，侯月梅1   

  1. 新疆医科大学第一附属医院，1心律失常VIP研究室，2医学研究中心实验动物科学研究部，新疆维吾尔自治区乌鲁木齐市 830054；   3解放军总医院肿瘤科，北京市 100853
  • 收稿日期:2010-12-20 修回日期:2011-02-19 出版日期:2011-05-14 发布日期:2011-05-14
  • 通讯作者: 侯月梅，博士，教授，博士生导师，新疆医科大学第一附属医院心律失常VIP研究室，新疆维吾尔自治区乌鲁木齐市 830054 houyuemei@ sina.com 并列通讯作者：时利，解放军总医院肿瘤科，北京市 100853
  • 作者简介:张玲☆，女，1982年生，博士，主要从事心律失常诊断、治疗及其机制的研究。 ydzhangling@ 126.com 并列第一作者：段明军，主要从事心律失常诊断、治疗及其机制的研究。
  • 基金资助:

    新疆维吾尔自治区高技术研究发展计划项目(201010105)，皮肤细胞诱导产生干细胞转基因构建心脏生物起搏器的动物实验研究。新疆维吾尔自治区重点实验室新疆心血管病重点实验室开放课题(XJDX0903-2009-03)，转SERCA2a基因治疗急性心肌梗塞电机械匹配的实验研究。

Isolation, culture and identification of ventricular cardiomyocytes from embryonal, neonate and adult C57 mice

Zhang Ling1, Duan Ming-jun2, Wei Qin2, Chen Hua1, Shi Li3, Hou Yue-mei1   

  1. 1Arrhythmia VIP Laboratory, 2Section of Laboratory Animal Research, the First Affiliated Hospital of Xinjiang Medical University, Urumqi  830054, Xinjiang Uighur Autonomous Region, China; 3Department of Tumor, General Hospital of Chinese PLA, Beijing  100853, China
  • Received:2010-12-20 Revised:2011-02-19 Online:2011-05-14 Published:2011-05-14
  • Contact: Hou Yue-mei, Doctor, Professor, Doctoral supervisor, Arrhythmia VIP Laboratory, the first affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uyghur Autonomous Region, China houyuemei@sina.com Correspondence to: Shi Li, Department of Tumor, General Hospital of Chinese PLA, Beijing 100853, China
  • About author:Zhang Ling☆, Doctor, Arrhythmia VIP Laboratory, the first affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uighur Autonomous Region, China ydzhangling@126. com Duan Ming-jun, Section of Laboratory Animal Research, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uighur Autonomous Region, China Zhang Ling and Duan Ming-jun contributed equally to this paper.
  • Supported by:

    the High Technology Research and Development Program of Xinjiang Uighur Autonomous Region, No. 201010105*; the Open Subject of Key Laboratory of
    Angiocardiopathy of Xinjiang Uighur Autonomous Region, No. XJDX0903-2009- 03*

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被引次数

2

摘要：

背景：培养的心肌细胞被广泛应用于心肌细胞的生理特性、毒性实验、基因工程、疾病模型和药物筛选等方面的研究。获得纯度较高活性良好的品系小鼠心肌细胞是研究的关键前提。
目的：分离和培养C57小鼠胎鼠、乳鼠及成年小鼠心室肌细胞。
方法：应用机械切碎心室肌后，胰蛋白酶消化不同发育阶段的C57小鼠心室肌细胞，差速贴壁1 h纯化心室肌细胞，锥虫蓝染色判定心肌细胞活力，体外分别培养48~72 h后分别行倒置显微镜、扫描及透射电镜观察细胞形态，微电极阵列评价细胞电生理指标，免疫组化鉴定。
结果与结论：经3~6次消化后，心室组织消化完全，即刻细胞存活率大于85%。倒置显微镜下观察，细胞呈梭形、多角形。12 h有少部分细胞搏动，48 h细胞交织成网，搏动呈同步性，搏动频率30~90次/min。说明用胰蛋白酶组织消化法可以成功地分离、培养，并获得形态、活力良好的胎鼠、乳鼠及成年C57小鼠心室肌细胞。

关键词: 心室肌细胞, 分离培养, 免疫组化, 电镜, 微电极阵列

Abstract:

BACKGROUND: Cultured cardiomyocytes are widely utilized in related researches such as physiological and toxicologic experiments, gene engineering, diseases model, drug screening and so on. How to harvest mouse cardiomyocytes with high activity is a key premise for these studies. 
OBJECTIVE: To establish a method to isolate and culture ventricular cardiomyocytes from embryonal, neonate and adult C57 mice.
METHODS: The ventricular myocardium from fetal, neonatal and adult C57 mice were minced and digested in trypsin, the purified ventricular cardiocytes were obtained by differential adhesion about 1 hour. The survival rate was assessed by trypan staining. Morphology of ventricular cardiomyocytes was observed through the methods of inverted phase contrast microscope, transmission electron microscope, scanning electron microscope, and the electrophysiological properties were identified through immunocytochemistry and microelectrode array.
RESULTS AND CONCLUSION: The ventricular tissues were almost completely digested through 3-6 times. Trypan staining showed that the survival rate of the cardiocytes cultured was more than 85%. Under the inverted phase contrast microscope, we found that the cells were fusiform or polyhedron and connected to each other and some cells began beat after 12 hours. The cells monolayer formed a network and beat spontaneously at the 30-90 times per minutes. The ventricular cardiomyocytes from embryonal, neonate and adult C57 mice can be obtained with well morphology and spontaneous beating with trypsin digestion.

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