Studies on Interactions of Flectric Field and Biomembranes

本研究计划的主要目的是（1）阐明电穿孔、电泳和电融合的基本机制，以及（2）开发用于细胞操作的标准应用程序，如基因转移和膜融合。本文还对电场对各种细胞现象的影响进行了研究，主要结果如下：（1）建立了脉冲激光显微镜，可直接观察细胞膜和脂质体上的电穿孔现象。获得了微秒级的时间分辨率和微米级的空间分辨率。结果表明，当膜电位超过阈值时，电穿孔在微秒内发生。已经观察到脂质体形成了意想不到的大孔（直径为微米）。(2)研制了高性能的电穿孔和电融合电源。(3)应用双向电泳和高压电场脉冲诱导生物膜囊泡和细胞的膜融合。(4)发现细胞内的巯基蛋白酶在细胞的电融合中起重要作用。(5)毫摩尔浓度的Ca^2+和Mg^2+促进了电场形成的孔的闭合。(6)建立了一种简单有效的哺乳动物细胞基因转移电穿孔方法。(7)产电离子泵如H^+-ATP酶和Ca^<2+>-ATP酶被整合到平面脂质双层中。这种重组膜提供了理想的系统，用于直接演示离子泵的产电性和电场效应，以及用于离子传输的动力学分析。(8)考察了表面电荷对膜形成的影响。

The main objects of this research project are (1) the elucidation of basic mechanisms of electroporation, diefectrophoresis and electrofusion, and (2) the development of standard application procedures for cell manipulation, such as gene transfer and membrane fusion. Investigations of electric field effects on various cellular phenomena, that were not yet studied, were also initiated.Major results obtained are as follows: (1) Pulse laser microscopy has been developed to observe directly electroporation on cellular membranes and liposomes. Time resolution of microseconds and spatial resolution of micrometers were obtained. Findings obtained shows that the electroporation takes place within a microsecond when membrane potential exceeds a threshold. The formation of unexpectedly large pores (micrometers in diameter) has been observed with liposomes. (2) High-performance power supply for electroporation and electrofusion was developed. (3) Diectrophoresis and high leictric field pulse were applied to induce membrane fusion in biomembrane vesicies as well as cells. (4) Intrinsic thiol proteases were found to take an important role in electrofusion of cells. (5) Millimolar Ca^<2+> and Mg^<2+> enhances the closing of pores formed by electric field. (6) A simple and efficient method of electroporation for gene transfer in mammalian cells was established. (7) Electrogenic ion pumps such as H^+- ATPase and Ca^<2+>-ATPase were incorporated into a planar lipid bilayer. This reconstituted membrane provides for ideal system for direct demonstration of the electrogenicity of ion pumps and the electric field effects, and for kinetic analysis of ion transport. (8) The effect of surface electric charge on membrane formation was examined.