模拟缺血再灌注对乳兔窦房结细胞起搏电流的影响

摘要

Objective To determine the effect of simulated ischemia/reperfusion (I/R) on the morphology and funny current(If) in sinoatrial node cells(SNCs) of newborn rabbits, and clarify the electrophysiological characteristic changes of SNCs after ischemia-reperfusion injury. Methods SNCs were isolated from newborn New Zealand rabbits by double enzyme hydrolysis/differential anchoring velocity, and then cultured under simulated ischemia reperfusion damage(95%N2+5%CO2 for 1h followed by 95%O2+5%CO2 for 3h). The cells cultured under normal condition(95%O2+5%CO2) served as normal control. The morphological changes of the cells were observed under inverted microscope. Patch-clamp technique was used to record action potential and the If. Results Normal SNCs were mainly spindle in shape and smooth and complete in surface, with fast frequency of pulsation. While, the SNCs after simulated I/R injury showed swelling and distortion, with granular and unsmooth cell membrane and retraction of pseudopodia. After simulated reperfusion, the maximum diastolic potential of If in SNCs was changed from (-50.9±5.3)mV to (-43.5±4.6)mV, current density was changed from (-3.2±0.41)pA/pF to (-2.47±0.32)pA/pF, with its steady-state activation curve obvious to the right when compared with that of control, and the half activating voltage was changed from (-98.6±2.3)mV to (-107.8±4.7)mV (P<0.05). Conclusion Simulated I/R induces injury in SNCs. Cell electrophysiological characteristics are changed, mainly in the If reconstruction.%目的：观察模拟缺血再灌注（I/R）对乳兔窦房结细胞形态及起搏电流（If）的影响，阐明I/R制备窦房结细胞损伤的电生理特征改变。方法选用新生新西兰乳兔，采用双酶解法/差速贴壁法分离窦房结细胞，模拟I/R制备受损模型，利用膜片钳技术记录动作电位和If。结果乳兔窦房结细胞主要呈梭形，搏动频率快，细胞表面光滑完整。模拟I/R后大部分细胞肿胀变形，伪足回缩，胞膜有较多颗粒、欠光滑。模拟再灌注后窦房结细胞If最大舒张电位由（-50.9±5.3）mV变为（-43.5±4.6）mV，电流密度由（-3.2±0.41）pA/pF变为（-2.47±0.32）pA/pF，其稳态激活曲线较对照组明显右移，半数激活电压由（-98.6±2.3）mV变为（-107.8±4.7）mV（P＜0.05）。结论模拟I/R导致窦房结细胞损伤，细胞电生理发生改变，尤以If重构为主。