4℃预处理对人中性粒细胞膜电流和极性的影响

Effect of 4℃ pretreatment on the membrane current and cell polarity in human neutrophils

对急性分离的人中性粒细胞采用4℃预处理是进行膜片钳实验前经常采取的步骤,但这一步骤对电生理记录结果有何影响尚无文献报道。本实验探讨这一步骤对电生理记录过程和实验结果的影响。结果显示,4℃预处理可以显著提高细胞的封接率,有利于对中性粒细胞进行电生理记录;封接率提高的原因与4℃预处理降低细胞的极性活动有关,但记录到的电压依赖性钾通道全细胞电流和大电导Ca2+依赖性K+单通道电流动力学没有显著的变化。这些结果表明,4℃预处理可能影响到细胞膜上与极性有关的脂膜变化,但对细胞膜上蛋白的功能影响较少。

To investigate the role of ion channels in the coupling responses of neutrophils to extracellular stimulus, it is necessary to study the membrane ion channel activities using patch-clamp technique. However, little has been known about the ion channel activities in neutrophils due to the difficulties in forming giga-seal with pipettes because of small diameter of neutrophils and the easily developed polarization. Some studies indicated that favorable results could be achieved through pretreatment at low temperature before electrophysiological recordings. But it remains unclear whether the pretreatment affects the membrane current and why the seal rate increases after low temperature pretreatment. The purpose of this study was to investigate the effects of 4℃pretreatment on the membrane current and cell polarity in human neutrophils. In the experiments, human neutrophils were isolated from fresh peripheral blood of healthy volunteers and divided into two groups （room temperature group and 4 ℃ pretreatment group）. Voltage-dependent K^＋ （Kv） currents were recorded in whole-cell voltage-clamp mode and large-conductance Ca^2＋-activated K^＋ （BKCa） currents were recorded using inside-out patches. The results showed that 4 ℃ pretreatment significantly inhibited cell polarity （P〈0.05）, and it took more time for neutrophils to form a polarity-cycle [（534±32） s, n=20] compared with those at room temperature [（257±24） s, n=20]. Meanwhile, seal rate significantly increased in 4 ℃ pretreatment group （64%） compared with that in the room temperature group （27.5%）. The seal rate and cell polarity rate during 0-1 min after 4℃ pretreatment were significantly different from those at room temperature, while no significant difference was found during 9-10 min between the two groups. Our results suggest that 4℃pretreatment can inhibit cell polarity and increase seal rate, but has no effects on membrane currents. It is also suggested that 0-1 min after 4℃ pretreatment is a more suitable time for electrophysiological recording in neutrophils.