细胞外液成分对耐钙心肌细胞急性分离及电生理记录的影响  

Influence of the ingredients in extracellular superfusate on acute isolation of calcium-tolerant ventricular myocytes and electrophysiological recordings

作　　者：周彤[1,2] 毛晓波[1] 毛奕[1] 柳强妮[3] 曾秋棠[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心内科,华中科技大学协和医院心血管病研究所,武汉430022 [2]内蒙古医学院第三附属医院心内科 [3]华中科技大学同济医学院药理系

出　　处：《山西医科大学学报》2009年第5期393-397,477,共6页Journal of Shanxi Medical University

基　　金：湖北省重大科技攻关基金资助项目(2006AA301A04)

摘　　要：目的探讨细胞外液成分对获取适于电生理研究的耐钙心室肌细胞及对L-型钙通道电流(ICa-L)、延迟整流钾电流(IK)记录的影响。方法家兔心室肌细胞急性分离采用Langendorff灌流装置及酶解分离技术,心脏灌流前实验组细胞外液为正常台氏液,对照组细胞外液为无钙台氏液,观察细胞外液中的钙离子对获取适于电生理记录的单个耐钙心肌细胞的影响;应用全细胞膜片钳技术,实验组分别以含BaCl2的台氏液及N-甲基-D-葡萄糖胺(NMDG)溶液作为细胞外液记录ICa-L及IK,对照组为正常台氏液。结果与对照组相比较,实验组心室肌细胞存活率和耐钙心肌细胞存活率均明显升高(71%±9.7%vs55%±10%;52%±7.9%vs39%±11%,n=10,P<0.05);实验组引出的ICa-L峰值电流大于对照组,且幅值稳定;实验组IK时间依赖性外向电流及尾电流随去极化时间延长增大作用更明显(1.39±0.05pA/pFvs0.53±0.14pA/pF;0.74±0.09pA/pFvs0.39±0.13pA/pF,n=10,P<0.05)。结论细胞外液中的钙离子有助于获取耐钙心肌细胞,以含BaCl2的台氏液及NMDG溶液作为细胞外液易于获得典型而稳定的ICa-L及IK。Objective To explore the influences of ingredients of extracellular superfusate on acute isolation of calcium-tolerant ventrieular myoeytes and recording on delayed rectifier potassium （ I K ） and L-type calcium current （ ICa-L ）. Methods Single ventricular myocytes of rabbits were obtained enzymatieally by retrograde Langendorff perfusing apparatus. Before Langendorff perfusion, the extracellular superfusate was Tyrode＇ s solution in experimental group and calcium-free Tyrode＇ s solution in control group. The effect of extracellular superfusate Ca^2＋ on calcium-tolerant ventrieular myocytes available for eleetrophysiological recordings were investigated. Whole-cell patch clamp recording technique was used to record the changes of I K and ICa-L following the administration of extracellular superfusate with BaCl2 and N-methyl-D-glucamine（NMDG） in experimental group and extracellular Tyrode＇ s solution in control group. Results The survival rates of ventricular myocytes and calcium-tolerant myocytes were significantly higher in experimental group than in control group（71% ±9.7% vs 55% ±10%,52% ± 7.9% vs 39%± 11% ,n = 10 ,P 〈 0.05 ）. In experimental group ,peak I Ca-L was elicited stably and larger than in control group. The time-dependent outward current and tail current of IK were higher in experimental group than in control group（1.39 ±0.05 pA/pF vs 0. 53 ±0.14 pA/pF,0.74 ±0.09 pA/pF vs 0. 39 ±0. 13 pA/pF,n = 10,P 〈 0. 05）. Conclusion Extracellular superfusate Ca^2＋ is helpful for isolating single calcium-tolerant cardiomyocytes. With BaCl2 and NMDG in extracellular superfusate, the ionic currents of ICa-L and IK can be elicited typically and easily.

关 键 词：膜片钳 耐钙心室肌细胞 L-型钙通道电流( I Ca-L ) 延迟整流钾电流( I K) 

分 类 号：R331.38[医药卫生—人体生理学]