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密度梯度离心法同时分离新生大鼠原代心肌细胞与成纤维细胞

陈希, 许瑞, 蒋易楠, 祝伟娜, 王耀辉

体药物河南省工程实验室，河南省抗体药物国际联合实验室，河南大学医学院细胞与分子免疫重点实验室，开封 475004；郑州大学基础医学院，郑州 450001

摘要

本文旨在探讨一种改良的同时分离新生大鼠原代心肌细胞和成纤维细胞的方法，以建立良好的原代心肌细胞及成纤维细胞研究模型。无菌状态下取Wistar乳鼠(出生不超过2天)心室，用II型胶原酶消化，控制消化时间及次数、搅拌速度、离心次数和速度等，结合Percoll密度梯度离心法分离心肌细胞及成纤维细胞，进行体外培养，观察细胞形态，继而用0.2%台盼蓝染色检测细胞存活率，免疫荧光染色法检测心肌肌钙蛋白I (cardiac troponin I, cTnI)、波形蛋白(Vimentin)及α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)的表达，分别用于鉴定心肌细胞和成纤维细胞纯度。结果显示，本方法5次分离的心肌细胞平均存活率达92%，纯度达95%以上，细胞生长状态良好，可见贴壁自发搏动；成纤维细胞平均存活率达96%，纯度达94%。本方法能同时获得新生大鼠原代心肌细胞和成纤维细胞，具有很高的产量、存活率及纯度，且操作简便，耗时短，重复性好，是一种较为理想的原代细胞分离、培养方法，可满足各种后续实验要求。

关键词： 心肌细胞; 成纤维细胞; 原代培养; 新生大鼠; Percoll分离

[Simultaneous separation of primary cardiomyocytes and cardiac fibroblasts from neonatal rats with density gradient centrifugation.] [Article in Chinese]

CHEN Xi, XU Rui, JIANG Yi-Nan, ZHU Wei-Na, WANG Yao-Hui

Henan Key Laboratory of Engineering Antibody Medicine, Henan International United Laboratory of Antibody Medicine, Key Laboratory of Cellular and Molecular Immunology, Henan University School of Medicine, Kaifeng 475004, China； Basic Medical of Collage, Zhengzhou University, Zhengzhou 450001, China

Abstract

To improve a fast and high-quality isolation method for culturing the primary cardiomyocyte and fibroblast in vitro, the neonatal Wistar rats were decapitated accordingly and left ventricles were isolated under the sterile condition. The ventricles were chopped and digested in the enzyme solution containing 0.5 mg/mL type II collagenase. During this process, the digesting time, frequency and stirring speed, centrifuging frequency and speed were strictly controlled. The cardiomyocytes were separated from the cardiac fibroblast by using the Percoll density gradient centrifugation. The cell viability was tested by staining with 0.2% trypan blue. The purity of cardiomyocytes and fibroblasts were determined by immunoflourescent staining with anti-cTnI, anti-Vimentin and anti-α-SMA antibodies. The results indicated that with this protocol, the viability and purity of cardiomyocytes were 92% and 95%. The automobile pulse of the adhered cardiomyocyte was visible. For fibroblasts, the cell viability and purity were 96% and 94%. Our results demonstrate that this advanced isolation method is reproducible, and can simultaneously produce high-quality primary cardiomyocytes and fibroblasts for the future study.

Key words: Cardiomyocyte; cardiac fibroblast; primary culture; neonatal rats; Percoll isolation

收稿日期：2015-02-09　　录用日期：2015-05-07

通讯作者：王耀辉　　E-mail: wangyaohui2001@sina.com

引用本文：

陈希, 许瑞, 蒋易楠, 祝伟娜, 王耀辉. 密度梯度离心法同时分离新生大鼠原代心肌细胞与成纤维细胞[J]. 生理学报 2015; 67 (4): 423-430.

CHEN Xi, XU Rui, JIANG Yi-Nan, ZHU Wei-Na, WANG Yao-Hui. [Simultaneous separation of primary cardiomyocytes and cardiac fibroblasts from neonatal rats with density gradient centrifugation.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (4): 423-430 (in Chinese with English abstract).