Pucihar Gorazd, Kotnik Tadej, Miklavcic Damijan, Teissié Justin
University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia.
Biophys J. 2008 Sep 15;95(6):2837-48. doi: 10.1529/biophysj.108.135541. Epub 2008 Jun 6.
The transport of propidium iodide into electropermeabilized Chinese hamster ovary cells was monitored with a photomultiplier tube during and after the electric pulse. The influence of pulse amplitude and duration on the transport kinetics was investigated with time resolutions from 200 ns to 4 ms in intervals from 400 micros to 8 s. The transport became detectable as early as 60 micros after the start of the pulse, continued for tens of seconds after the pulse, and was faster and larger for higher pulse amplitudes and/or longer pulse durations. With fixed pulse parameters, transport into confluent monolayers of cells was slower than transport into suspended cells. Different time courses of fluorescence increase were observed during and at various times after the pulse, reflecting different transport mechanisms and ongoing membrane resealing. The data were compared to theoretical predictions of the Nernst-Planck equation. After a delay of 60 micros, the time course of fluorescence during the pulse was approximately linear, supporting a mainly electrophoretic solution of the Nernst-Planck equation. The time course after the pulse agreed with diffusional solution of the Nernst-Planck equation if the membrane resealing was assumed to consist of three distinct components, with time constants in the range of tens of microseconds, hundreds of microseconds, and tens of seconds, respectively.
在电脉冲期间及之后，用光电倍增管监测碘化丙啶进入电穿孔的中国仓鼠卵巢细胞的转运情况。以400微秒至8秒的时间间隔，时间分辨率从200纳秒至4毫秒，研究了脉冲幅度和持续时间对转运动力学的影响。早在脉冲开始后60微秒时转运就可被检测到，在脉冲后持续数十秒，并且对于更高的脉冲幅度和/或更长的脉冲持续时间，转运更快且量更大。在固定脉冲参数下，转运进入汇合的单层细胞比转运进入悬浮细胞慢。在脉冲期间及脉冲后的不同时间观察到荧光增加的不同时间进程，反映了不同的转运机制和正在进行的细胞膜重封过程。将数据与能斯特 - 普朗克方程的理论预测进行比较。在延迟60微秒后，脉冲期间荧光的时间进程近似线性，支持能斯特 - 普朗克方程的主要电泳解。如果假设细胞膜重封由三个不同的成分组成，时间常数分别在数十微秒、数百微秒和数十秒范围内，则脉冲后的时间进程与能斯特 - 普朗克方程的扩散解相符。
