成年大鼠心房肌细胞分离技术的优化及L型钙电流记录  

Optimizing techniques for isolating adult rat atrial myocytes and recording calcium currentss

作　　者：梁俊卿 汤宝鹏[1,2] 沈俊 曼则热姆·热杰普 郭衍楷 张玲[1,2] Liang Junqing;Tang Baopeng;Shen Jun;Manzeremu Rejiepu;Guo Yankai;Zhang Ling(Xinjiang Key Laboratory of Cardiac Electrophysiology and Cardiac Remodeling,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang,China;Cardiac Pacing and Electrophysiology Department,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang,China)

机构地区：[1]新疆医科大学第一附属医院心电生理与心脏重塑重点实验室,新疆乌鲁木齐830054 [2]新疆医科大学第一附属医院心脏起搏电生理科,新疆乌鲁木齐830054

出　　处：《中国心脏起搏与心电生理杂志》2024年第5期349-355,共7页Chinese Journal of Cardiac Pacing and Electrophysiology

基　　金：国家自然科学基金(82200360);省部共建中亚高发病成因与防治国家重点实验室开放课题资助项目(SKL-HIDCA-2022-XXG4)。

摘　　要：目的 本研究旨在探索并优化一种稳定有效的成年大鼠心房肌细胞分离方法,以便于进行膜片钳电生理实验。方法 采用先结扎上腔静脉和肺静脉,随后通过主动脉逆行插管进行Langendorff灌流,使用胶原酶Ⅱ进行约27 min的反复灌流消化,继而从心房组织中剪取心肌组织,在室温下用KB液剪碎并轻轻吹打以分离细胞。随后通过逐步复钙法恢复细胞中的钙离子浓度,并在室温下静置1 h后,进行膜片钳实验。结果 成功分离出约(7.5±1.4)×10^(6)(n=6)个原代心房肌细胞,其中绝大多数细胞呈现典型的长杆状形态,并且在外加钙离子浓度恢复至1 mmol/L后,约80%的细胞能够保持其形态稳定。成年大鼠心房肌细胞的动作电位表现为快速复极的尖锐三角形。结论 本研究成功建立了一种稳定的成年大鼠心房肌细胞分离技术,为进一步深入研究心房肌细胞的电生理行为提供了可靠的实验材料。Objective This study aimed to develop and refine a robust method for isolating adult rat atrial myo-cytes,facilitating electrophysiological analyses via the patch-clamp technique.Methods A meticulous procedure involving the ligation of both the superior vena cava and the pulmonary vein,followed by Langendorff perfusion through retrograde aortic cannulation,was employed.Collagenase II was used for repeated perfusion digestion over approximately 27 minutes.Atrial tissues were excised,minced,and gently triturated in KB solution at room tem-perature to dissociate the cells.The cellular calcium ion concentration was subsequently normalized through a careful stepwise recalcification process.After allowing the cells to settle at room temperature for an hour,patch-clamp ex-periments were conducted.Results The protocol resulted in the isolation of approximately(7.5±1.4)×10^(6)(n=6)primary atrial myocytes,predominantly exhibiting the characteristic elongated rod-like morphology.Notably,a-round 80%of these cells retained their structural integrity after recalcification to a 1 mmol/L external calcium ion concentration.The action potentials recorded from these myocytes displayed a sharply triangular shape characteristic of rapid repolarization phases.Conclusion The study successfully established a reliable and efficient protocol for i-solating adult rat atrial myocytes.This method provides a solid foundation for conducting detailed electrophysiological studies on atrial myocytes,offering valuable insights into their functional properties.

关 键 词：心血管病学 成年大鼠 心房肌细胞 分离 膜片钳 

分 类 号：R331.38[医药卫生—人体生理学]