成年大鼠心肌细胞分离方法的改良

A modified method for isolation of adult rat cardiomyocytes

背景:目前多采用酶解法分离成年大鼠心肌细胞,但在灌流装置、灌流液体和灌流方法等方面的差异较大,且多以单个细胞的急性分离为目的,仅适于进行电生理研究。目的:建立稳定的改良成年SD大鼠心肌细胞分离方法,使之适于进行原代培养和研究。设计、时间及地点:细胞学体外实验,于2008-03/09在华中科技大学同济医学院附属同济医院分子心脏病中心完成。材料:8～10周龄雄性SD大鼠30只,由同济医学院实验动物中心提供,Ⅱ型胶原酶、蛋白酶和不含脂肪酸的牛血清白蛋白分别为美国Worthington公司、美国Sigma公司和德国Merck公司产品。方法:SD大鼠麻醉后,快速取出心脏,采用改良的Langendorff灌流装置行主动脉逆行灌注,无钙Krebs-Henseleit缓冲液(KH液)非循环灌流4min,0.6g/LWorthington2型胶原酶联合0.03g/L蛋白酶消化约15min,剪下心室组织于消化液和10g/L牛血清白蛋白的混合液中,剪碎后于37℃振摇孵育10min,200μm尼龙网过滤,500g离心1min,梯度复钙,自然沉降。主要观察指标:心肌细胞的形态、活力和产量。结果:复钙后的杆状存活心肌细胞得率为(73±5.6)%(n=30),其中90%以上的杆状心肌无明显搏动,横纹清晰。每只大鼠心脏的平均存活心肌细胞产量为(4.2±0.4)×106。结论:经过改良的Langendorff逆行主动脉灌流酶解法分离成年大鼠心肌细胞,可稳定获得高比例的存活心肌细胞并用于原代培养和细胞/分子生物学研究。

At present, adult rat cardiomyocytes are usually isolated by enzymolysis method with different kinds of perfusion apparatuses, collagenases and methods. It is mainly aimed at emergent isolation of single cardiomyocyte and only appropriate for electrophysiological study. OBJECTIVE： To develop and optimize a stable adult Sprague-Dauley rat cardiomyocytes isolation method for primary culture and research. DESIGN, TIME AND SETTING： The cytology in vitro experiment was performed at the Molecular Cardiology Center, Department of Cardiology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from March to September 2008. MATERIALS： Thirty male Sprague-Dawley rats aged 8 to 10 weeks were obtained from the Laboratory Animal Center, Tongji Medical College. Collagenase type Ⅱ, protease and bovine serum albumin （BSA） without fatty acid were purchased from Worthington Biochemical Corporation, Sigma Corporation and Merck Corporation, respectively. METHODS： After successful anesthesia, the rat heart was quickly excised out with sufficient length of the aorta and perfused by modified Langendorff system： firstly perfusion with calcium-free Krebs-Henseleit buffer （KHB） in a non-recirculating manner for 4 minutes, and then digestion with 0.06% collagenase （Worthington, type 2） and 0.003% protease for about 15 minutes, thereafter ventricular tissue was minced and incubated in a digestion medium with 10 g/L BSA at 37 ℃ for 10 minutes. After filtered with 200 μm nylon net and centrifuged at 500 g for 1 minute, the isolated cells were performed gradient calcium reintroduction and allowed to natural sedimentation. MAIN OUTCOME MEASURES： Morphology, vitality and yield of isolated rat cardiomyocytes. RESULTS： A large amount of rod-shaped rat cardiomyocytes with clear cross striations and high activity [（73±5.6）%, n=30] were acquired after calcium reintroduction, more than 90% of rod-shaped cardiomyocytes were quiescent with clear transverse striation. The average yield of viable cardiomyocytes was （4.2±0.4）×106 per heart. CONCLUSION： A high yield of cardiomyocytes isolated by aorta retrograde perfusion with digestive enzymes using modified Langendorff system can be acquired stably and applicable for primary culture and cell or molecular biology study.