能量可控陡脉冲对肿瘤细胞内钙离子浓度及膜电位的影响

背景与目的：前期研究表明，陡脉冲电场对肿瘤细胞具有明显的杀伤作用，但其确切机制尚不清楚。本文拟探讨不同剂量陡脉冲对细胞内游离Ca^2＋浓度（[Ca^2＋]i）和细胞膜电位的影响。方法：将乳腺癌细胞MDA—MB-231分为对照组和5个不同剂量能量可控陡脉冲（energy controllable steep pulses，ECSP）处理组．分别用荧光探针Fluo-3／AM标记Ca^2＋、DiBAC4（3）标记细胞膜电位，应用激光共聚焦显微镜静态观察不同剂量ECSP处理后乳腺癌细胞内荧光探针的平均荧光强度，分析细胞内Ca^2＋浓度的变化以及细胞膜电位的改变。同时观察细胞胞外有或无钙离子时，ECSP对细胞内游离Ca^2＋浓度的影响。结果：静态观察发现在细胞受到能量较低电场刺激后，细胞外钙内流；当能量增大后，细胞内钙大量外流。实时动态观察发现在低能量的脉冲处理时。细胞内Ca^2＋的荧光强度和上升速率随脉冲电场强度的增加而增加：但当能量达到电压285V、频率100Hz时，荧光强度反而呈现明显的下降。在没有外Ca^2＋的情况下，细胞内Ca^2＋浓度的增加虽然与有外钙时相比显著下降，但在脉冲电场作用下在脉冲电场作用下仍缓慢上升。低剂量的ECSP可诱导细胞DiBAC4（3）的荧光强度增强，表明细胞发生去极化；但当剂量继续增加时，细胞DiBAC4（3）的荧光强度反而减弱。表明细胞发生超极化。结论：低能量陡脉冲可使细胞膜发生去极化，诱导细胞外钙离子内流；高能量陡脉冲可直接破坏细胞膜．使细胞膜发生超极化，诱导细胞内钙离子外流．导致肿瘤细胞发生坏死。

Effects of energy controllable steep pulses on intracellular calcium concentration and cell membrane potential

Background and Objective. Our previous experiments showed that steep pulses could kill tumor cells, but the mechanism is unclear. This study was to probe the effects of different dosages of energy controllable steep pulses （ECSP） on intracellular concentration of dissociative calcium ion （[Ca^2＋] i） and cell membrane potential. Methods.The breast carcinoma MDA- MB-231 cells were divided into control group and five ECSP （different dosages） groups, Ca^2＋ was labeled by Fluo-3/AM and cell membrane potential was labeled by DiBAC4（3）. The mean fluorescence intensity in MDA-MB-231 cells was observed by laser confocal microscopy after ECSP treatment, The changes of calcium concentration and cell membrane potential after ECSP treatment were analyzed. The changes of intracellular [Ca^2＋]i after ECSP treatment were also observed either with or without Ca^2＋ outside of the cells. Results： Ca^2＋ outflow was observed when the cells were treated with lower dosage of pulse in quiet state; the outflow was enhanced with the dosage increase. In real-time kinetic detection, intracellular Ca^2＋ concentration was increased with the increase of pulse electric field intensity when cells were treated with lower dosages of ECSP. When the voltage was 285 V, frequency was 100 Hz, [Ca^2＋]i decreased obviously. The intracellular Ca^2＋ concentration was obviously lower in the cells without outside Ca^2＋ than in cells with outside Ca^2＋, but it still increased gradually. Low dosage of ECSP induced the increase of cell inembrane potential, indicating the depolarization of cell membrane. With increase of the dosage, cell membrane potential was attenuated, indicating the superpolarization of cell membrane. Conclusion. Lower dosage of ECSP can induce the depolarization of cell membrane and the inflow of outside Ca^2 ＋ higher dosage of ECSP can directly destroy the cell membrane and induce the superpolarization of cell membrane, then induce the outflow of intracellular Ca^2＋ which causes the necrosis of tu