改良成年小鼠心室肌细胞分离方法  

The improvement of adult mouse cardiomyocytes isolation method

作　　者：石山慧 吕定超 翟旭雯[1] 李端端[1] 周密[1] 左琳[1] SHI Shan-hui LV Ding-choo ZHAI Xu-wen et al(Department of Physiology, School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, China)

机构地区：[1]山西医科大学基础医学院生理学系,山西省太原市030001

出　　处：《中国心血管病研究》2016年第8期748-751,766,共5页Chinese Journal of Cardiovascular Research

基　　金：国家自然科学基金青年科学基金项目（项目编号：81200120）;山西省留学回国留学人员科研资助项目（项目编号：2014-036）

摘　　要：目的 改进小鼠成体心肌细胞的分离方法,以提高小鼠心室肌细胞的成活率和收获量.方法 采用心脏在体主动脉插管,行独创的前加压灌流法冲洗心脏后接入改进的langendorff装置,用胶原酶液Ⅱ灌流、消化心脏后,将心脏剪碎并用吸管吹打,经200目滤网过滤后获得单个小鼠心室肌细胞.检测并对比在体和离体主动脉插管获得的心肌细胞的状态：梯度复钙后,在倒置显微镜下观察细胞的形态和杆状心室肌细胞的数目.结果 研究结果显示,改良的主动脉逆行灌流法在缩短手术时间的同时,提高了小鼠心室肌细胞收获量和存活率.本课题组利用医用三通管,独创倒置灌流排气法,可以迅速排空灌流装置中的气体;利用注射器和灌流针结合,独创前加压灌流法,可以快速彻底地冲洗心脏中的血液;并采用实验室常规眼科镊和血管钳巧妙组合成协助装置,可以单人独立完成插管,不需要助手协助,节约人力,节省经费开支.结论 成年小鼠心室肌细胞分离方法的改进使整个实验流程简化,缩短了实验时间,操作简便易学;同时该分离方法稳定、可靠、有效,可以为同类型实验提供思路和借鉴.Objective The mouse is a better experimental animal suitable for genetic study. In order to increase the harvest and survival rate of aduh mouse ventricular cardiomyoctyes AMVCMs, we try to improve the isolation method. Methods In the experiment, we used aortic cannulation in vivo and high-pressure perfusion to flush the heart, then collagenase Ⅱ was used to digest the heart by the Langendorff apparatus. Hereafter the fully digestion, the atria of the heart was removed. The remainder of the heart was minced. After repeated pipetting, cell suspension was filtered with 200μm nylon net, AMVCMs can be collected. The research has compared the amount and quality of AMVCMs between aortic cannulation in vivo and in vitro method after gradient calcium reintroduc- tion. Results The result showed that the improved aortic cannulation method can shorten the operation time and increase the harvest and survival rate of AMVCMs. Our group original create the function of inverted exhaust by using an medical three-way pipe to remove all traces^of the bubble in the perfusion apparatus; create pre-perfusion method by combining the syringe and perfusion needle to wash the heart rapidly and thoroughly; combine ophthalmology tweezers and forceps into an equipment to assist to realize cannulation by single person with saving resources and fund. Conclusion The improvements can simplify the whole experimental procedure, shorten the isolation time which is easy to learn. The improved method is stable, reliable and effective which can provide ideas for similar experiments.

关 键 词：成年小鼠 心室肌细胞 分离 LANGENDORFF 胶原酶 

分 类 号：Q95-33[生物学—动物学] R542.2[医药卫生—心血管疾病]