改良的成年SD大鼠心室肌细胞分离与培养方法  

A modified method for isolation and culture of ventricular myocytes in adult SD rats

作　　者：韩福平[1] 陈耀旭[1] 苏浩[1] 吕南英 殷春霞[1] 黄礼杰[1] 谭文[1] 

机构地区：[1]华南理工大学生物科学与工程学院/生物医药前孵化器研究中心/广东省发酵与酶工程重点实验室,广东广州510006

出　　处：《广东药学院学报》2015年第4期518-521,共4页Academic Journal of Guangdong College of Pharmacy

基　　金：广东省科技计划项目(2012B011000050);广州市重大专项科技项目(201300000051)

摘　　要：目的建立稳定高效的成年大鼠心室肌细胞分离与培养方法。方法成年SD大鼠麻醉后,迅速开胸取心,将主动脉连接于Langendorff装置上进行逆向灌流,用0.8 mg/m LⅡ型胶原酶和0.1 mg/m L蛋白酶双酶灌注消化分离心肌细胞,采用自然沉降法纯化心室肌细胞,一部分细胞用改良的M199培养基进行细胞培养,另一部分使用台盼蓝染色进行细胞活性检测并计数,同时使用激光共聚焦显微镜观察细胞收缩及钙瞬变。结果本方法可获得(81.9±2.2)%具有活性的成年大鼠心室肌细胞,每只SD大鼠心脏的细胞产量可达(2.0±0.1)×107个,电刺激时细胞同步稳定收缩。结论该分离和培养方法高效、稳定,分离出的细胞活性高,产量高,可用于原代培养。Objective To develop a stable method for isolation and culture of ventricular myocytes in adult Sprague-Dauley（ SD） rats. Methods Rat heart was removed quickly after anaesthetized and hanged on the Langendorff apparatus. Then the heart was perfused retrogradely from aorta and digested with collagenaseⅡ（ 0. 8 mg/m L） and protease（ 0. 1 mg/m L）. The acquired ventricular myocytes were purified with natural sedimentation and cultured with modified M199 medium. The viability and yield of the isolated cardiomyocytes were assessed by Typan Blue assay. The contractility and calcium transients were measured by confocal laser scanning microscope by electric stimulation. Results The percentage of survival ventricular myocytes was（ 81. 9 ± 2. 2） %,the number of ventricular myocytes was（ 2. 0 ± 0. 1 × 107） from every heart and the isolated cardiomycytes presented good contractility under electrical stimulation.Conclusion A stable isolation and culture method is established with high viability and yield for primary culture.

关 键 词：成年大鼠 心室肌细胞 分离 原代培养 

分 类 号：R542.2[医药卫生—心血管疾病]