细胞膜电穿孔现象机理研究
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摘要
近年来，人们对电磁场产生的生物学效应及其应用的研究越来越广泛，包括电磁波的生物热效应和非热效应．其中脉冲电磁场下非热生物效应是生物电磁学中最新的一个领域，普遍认为电磁脉冲非热效应的原发点为细胞膜，膜电穿孔是解释生物体中非热生物效应的关键。
电穿孔效应是指在适当的高压脉冲电场下，细胞或组织间起相对隔离作用的“屏障”内快速形成液态通道的现象，是电场与具有不同的介电常数而且易变形的物质相互作用的结果。
本文首先介绍电穿孔的机理及应用研究现状，并结合我们对细胞膜电穿孔基本特性的认识，设计制作了一台用于电穿孔研究用的新型脉沖电源，该电源具有双极性输出，其幅值、脉宽、重复频率及计数个数均可调节，文中对其系统结构及性能参数作了简要的介绍；论文中采用HepG2细胞作为靶细胞，进行了电穿孔电场阈值的实验研究；结合试验研究和理论分析，建立了基于细胞膜电应力的电穿孔模型，对细胞膜电穿孔过程进行了详细的描述；此外对电穿孔研究中出现的细胞内电处理效应，我们对其机理进行了初步的探索。
In the recent years, the biological effects of electromagnetic field and its applications have been widely studied including the thermal and non-thermal effects. Research on the non-thermal effects of pulsed electromagnetic field is a new field of bioelectromagnetics. It is usually believed that the origin of non-thermal effects of pulsed electromagnetic field is cell membrane and the electroporation of cell membrane is a key of explaining the mechanism of non-thermal.
The phenomenon of "electroporation" occurs if the cell was under an appropriate pulse field. Electroporation is believed to be the aqueous pathways which rapidly were created through lipid-containing barriers in cells and tissue. The driving force is the physical interaction between electric fields and two matters with different dielectric constants.
This thesis introduces the research and progress on the mechanism of electroporation and its applications. A pulsed power supply has been designed for research on electroporation which can output bipolar rectangular pulses, and can vary the voltage scope, pulse width and repetition .The principle system and specifications of this instrument are introduced in this paper. Using liver cancer cells as target cells, the threshold of field of electroporation is studied. Based on the experiments and analysis, an electrical stress model of electroproation has been set up and the process of electroporation has been described. Also we discuss the mechanism of the intracellular electro- manipulation .
电穿孔效应是指在适当的高压脉冲电场下，细胞或组织间起相对隔离作用的“屏障”内快速形成液态通道的现象，是电场与具有不同的介电常数而且易变形的物质相互作用的结果。
本文首先介绍电穿孔的机理及应用研究现状，并结合我们对细胞膜电穿孔基本特性的认识，设计制作了一台用于电穿孔研究用的新型脉沖电源，该电源具有双极性输出，其幅值、脉宽、重复频率及计数个数均可调节，文中对其系统结构及性能参数作了简要的介绍；论文中采用HepG2细胞作为靶细胞，进行了电穿孔电场阈值的实验研究；结合试验研究和理论分析，建立了基于细胞膜电应力的电穿孔模型，对细胞膜电穿孔过程进行了详细的描述；此外对电穿孔研究中出现的细胞内电处理效应，我们对其机理进行了初步的探索。
In the recent years, the biological effects of electromagnetic field and its applications have been widely studied including the thermal and non-thermal effects. Research on the non-thermal effects of pulsed electromagnetic field is a new field of bioelectromagnetics. It is usually believed that the origin of non-thermal effects of pulsed electromagnetic field is cell membrane and the electroporation of cell membrane is a key of explaining the mechanism of non-thermal.
The phenomenon of "electroporation" occurs if the cell was under an appropriate pulse field. Electroporation is believed to be the aqueous pathways which rapidly were created through lipid-containing barriers in cells and tissue. The driving force is the physical interaction between electric fields and two matters with different dielectric constants.
This thesis introduces the research and progress on the mechanism of electroporation and its applications. A pulsed power supply has been designed for research on electroporation which can output bipolar rectangular pulses, and can vary the voltage scope, pulse width and repetition .The principle system and specifications of this instrument are introduced in this paper. Using liver cancer cells as target cells, the threshold of field of electroporation is studied. Based on the experiments and analysis, an electrical stress model of electroproation has been set up and the process of electroporation has been described. Also we discuss the mechanism of the intracellular electro- manipulation .
引文
1．陈国璋，陈惠晓．谈谈生物电磁学研究热点——非热效应[J]．物理，1998，27(3)：151-155
2．宋婷，刘瑛岩．生物电磁学的研究进展[J]．华北电力大学学报，2002，第29卷增刊：1-5
3．黄卡玛，李颖等．近年来弱电磁场(波)生物效应机理研究进展[J]．中国医学物理学报，2000，17(1)：36-40
4．康刚等，分析脉冲电磁场对人体作用的一种人体简易色散模型，电子学报，1998年6月
5．王保义等．电磁场非热效应的机理研究，电子科技导报，1997年第二期，p5-8
6．王保义，刘长军，郭庆功等．电穿孔——解释生物体非热效应的关键机理[J]．四川大学学报(自然科学版) 1997，34(3)：310
7. Singer, S.J.,et al.,Science,1972,175:720-731
8. James C.Weaver. Electroporation in Cells and tissues[J]. IEEE Trans on plasma science, 2000,28(1):24-33
9. James C.Weaver, Electroporation of Biological Membranes from Multicellular to Nano Scales. Workshop on "Dielectric Properties and Electrical Breakdown of Biological Membranes", October 20,2002
10. U.Zimmermann, et al., Transcellular ion flow in Escherichia coli B and electrical sizing of bacterias, Biophys.J., 1973, 13(10):1005-1013
11. U.Zimmermann, et al., Dielectric breakdown of cell membranes, Biophy.J., 1974, 14(11):881-899
12. Mitsugi Senda, Induction of cell fusion of plant protoplasts by electrical stimulation, Plant and Cell Physiol., 1979,20(7):1441-1443
13. U.Zimmermann, et al., Electric field-induced cell to cell fusion, J.Memb.Biol., 1982, 67(3):165-182
14. U. Zimmermann, et al., Electric field-induced release of chloroplasts from plant protoplasts,Naturwissenschaften, 1982,69:451-452
15. M. Nishiguchi, et al., An improved method for electroporation in plant protoplasts:
infection of tobacco protoplasts by tobacco mosaic virus particles, Plant Cell Reports,1987, 6(2):90-93
16. Donald C.Change, et al, Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy, Biophys J. 1990, 58(1): 1-12
17．张弘，刘长军，王保义等．低强度瞬态电磁脉冲引发细胞膜电穿孔的试验初探．细胞生物学杂志．1999，22(1)
18．汪和睦，李金慧．细胞电穿孔导入DNA的动态特性．微生物学通报．1994，21(2)
19．刘缨，张锦珠．细胞电穿孔动态过程的荧光测量．生物物理学报．1994，10(2)
20．张锦珠，孔可健，脉冲电场对人红细胞膜结构影响的冷冻断裂研究，试验生物学报，1994，27(2)：183-191
21. R.Benz, U.Zimmermann, High electric field effects on the cell membranes of Halicystic parvula, Planta, 1981, 152(4):314-318
22. Dimiter S.Dimitrov, Rakesh K.Jain. Membrane Stability. Biochimica et Biophysica Acta. 1984,779:437-468
23. U.Zimmermann,Electric field-mediated fusion and related electrical phenolmena, Biochimica et Biophy.Acta,1982,694(3):227-277
24. Arthur E.Sowers, A long-lived fusogenic state is induced in Erythrocyte Ghosts by electric pulses, Journal of Cell Biol., 1986,102(4):1358-1362
25. W. Mehrle, et al.,Evidence for asymmetrical uptake of fluorescent dyes through electro-permeabilized membranes of avena mesophyll protoplasts. FEBS Letters, 1985,185-89-94
26. I.G. Abidor, Electric breakdown of bilayer lipid membranes: Ⅰ. The main experimental facts and their qualitative discussion,, 1979,6:37-52
27. Hermann Berg, et al., Possibilities of cell fusion and transformation by electrostimulation,Bioelectrochem.Bioenerget., 1984,12:119-133
28. Tadej Kotnik, Damjian Miklavi,Analytical Description of Transmembrane Voltage Induced by Electric Fields on Spheroidal Cells.Biophysical Journal,2000, 79:670-679
29. U.Zimmermann, G.Pilwat, et al., Effects of External Electrical Fields on Cell Membranes,Bioelectrochem.Bioenerg.,1976,3:58-83
30. Herzog, R., et al., Life Science, 1986, 39:2279-2288
31. Weaver J C. Electroporationin cells and tissues: A biophysical phenonmenon due to electromagmetic fields, Radio Science, 1995, 30(1): 205
32. Wong T K, Neumarme, Electric field mediated gene transferr, Biochem Biophys Res Commun, 1982,107:584-587
33. Karl H.Schoenbach, Sunao Katsuki, Robert H.Stark, E.Stephen Buescher, and Stephen J.Beebe, Bioelectrics-new applications for pulsed power technology[J], IEEE Trans on plasma science, 2002,30(1): 293-300.
34. Mouneimney, Electroinsertion of full length recombineant CD4 in to red blood cell membrane, Biochem. Biophys Acta,1990,1027:53-58
35. Mirlm, Electrochemotherapy: potentialtion of antitumore effect of bleomycin by local electric pulsed, Eur J Cancer, 1991,27:68-72
36．姚成果，孙才新．陡脉冲不可逆电击穿恶性肿瘤细胞的研究，高电压技术，2003，29(1)：45-47
37. Weaver JC.,Recent advances in skin electroporation: mechanism and efficacy.In:2nd World congress for EMBM Abstract Book, Bologna, Italy June 8-13,1994:44
38．张占松，蔡宣三等编著，开关电源的原理与设计，电子工业出版社，p1-3．
39．张芝涛等，脉宽调制型(PWM)高压电源的研究，电力环境保护，1994，10(1)：35-38
40．冯挹，30kHz高频开关电源变压器的设计，http://www.willar.com
41．张文利，高压大功率开关电源技术的研究(硕士论文)
42．李希茜，高频变压器的设计，现代电子技术，2001年第9期，p7-8
43．彭勇，王子淑等，低强度瞬态电磁场下动物细胞的电穿孔效应，四川大学学报，2000，37(4)：586-589
44．(美) F．奥斯伯等著，颜子颖，王海林译，精编分子生物学试验指南，北京，科学出版社，1998．6，pp793-800
45．王保义，杨杰斌等，毫微秒电磁脉冲的生物效应试验研究和机理分析，中国科学(C辑)，1997，27(1)：35-39
46．邹积岩，吴为民，脉冲电场食品处理技术，高电压技术，2000，26(6)：31-33
47．汪和睦，汪洲，细胞电穿孔及电融合的机理及应用，生物化学与生物物理进展，1994，21(4)：322-326
48．王保义，刘长军，电穿孔——解释生物体中非热生物效应的关键机理，四川大学学报，1997，34(3)：310-314
49. J.M.Crowley, Electrical breakdown of biomolecular lipid membranes as an electromechartical instability, Biophys.J., 1973,13:711-724
50．牛中奇，细胞膜上自电场应力，中国生物医学工程学报，1994，13(1)：90-95
51. D.S. DIMITROV, Electroporation and Electrofusion of Membranes,. Handbook of Biological Physics, Volume 1.edited by R. Lipowsky and E. Sackmann.
52. Barry H.Honig, Electrostatic interactions in membranes and proteins. Ann. Rev. Biophys. Chem., 1986,15:163-93
53. J.A.Stratton, Electromagnetic Theory, McGraw-Hill Book Company Inc., New York,NY,USA,1941.
54. I.Krakovsky, T.Romijn, et al.,A few remarks on the electrostriction of elastomers, Journal of applied physics, 1999,85(1):628-629
55. T.J.Lewis, Electromechanical effects in biological membrane,2002 Annual report conference on electrical insulation and dielectric phenomena, pp87-90
56. J.N.Israelachvili, S.Marcelja and R.G.Horn, Physical Principles of Membrane Organization, Quarterly Rev. of Biophys., 1980,13:121-200
57. J.N.Israelachvili, Intermolecular and Surface Forces,2nd Edition, Academic Press, London
58. Stephen J.Beebe, et al., Nanosencond Pulsed Electric Field(nsPEF) Effects on Cells and Tissues: Apoptosis Induction and Tumor Growth Inhibition, IEEE transactions on plasma science,2002, 30(1): 286-292
59. R.P.Joshi,Q.Hu and K.H.Schoenbach, Dynamical Modeling of Cellular Response to Short High-Intensity Electric Fields, Workshop on "Dielectric Properties and Electrical Breakdown of Biological Membranes", October 20,2002
60. Karl H.Schoenbach, The effect of pulsed electric fields on biological cells: experiments and applications,IEEE Trans on plasma science, 1997,25(2):284-292.
61. Karl H.Schoenbach, Stephen J.Beebe, and E.Stephen Buescher. Intracellular effect of
ultrashort electrical pulses[J], Bioelectromagnetics, 2001,22(6):440—448
62. Martin Garderse. Utrashort pulse electroporation: application of high pulsed electric fields to induced caspase activation of human lymphocytes[J], IEEE Trans on plasma science, 2000, 28(2): 667-670.
63. T.Kotnik, F.Bobanovic, D.Miklavacic, "Sensitivity of transmembrane voltage induced by applied electric fields-a theoretical analysis", Bioelectrochem. Bioenerg. 1997, 43: 285-291.
64. Tadej Kotnik, Damijan Miklavi. Theoretical Evaluation of the Distributed Power Dissipation in Biological Cells Exposed to Electric Fields[J]. Bioelectromagnetics, 2000,21:385-394
65. Kenneth R. Foster. Thermal and nonthermal mechanisms of interaction of radio-frequency energy with biological systems, IEEE Trans on plasma science, 2000, 28(1): 15-23
2．宋婷，刘瑛岩．生物电磁学的研究进展[J]．华北电力大学学报，2002，第29卷增刊：1-5
3．黄卡玛，李颖等．近年来弱电磁场(波)生物效应机理研究进展[J]．中国医学物理学报，2000，17(1)：36-40
4．康刚等，分析脉冲电磁场对人体作用的一种人体简易色散模型，电子学报，1998年6月
5．王保义等．电磁场非热效应的机理研究，电子科技导报，1997年第二期，p5-8
6．王保义，刘长军，郭庆功等．电穿孔——解释生物体非热效应的关键机理[J]．四川大学学报(自然科学版) 1997，34(3)：310
7. Singer, S.J.,et al.,Science,1972,175:720-731
8. James C.Weaver. Electroporation in Cells and tissues[J]. IEEE Trans on plasma science, 2000,28(1):24-33
9. James C.Weaver, Electroporation of Biological Membranes from Multicellular to Nano Scales. Workshop on "Dielectric Properties and Electrical Breakdown of Biological Membranes", October 20,2002
10. U.Zimmermann, et al., Transcellular ion flow in Escherichia coli B and electrical sizing of bacterias, Biophys.J., 1973, 13(10):1005-1013
11. U.Zimmermann, et al., Dielectric breakdown of cell membranes, Biophy.J., 1974, 14(11):881-899
12. Mitsugi Senda, Induction of cell fusion of plant protoplasts by electrical stimulation, Plant and Cell Physiol., 1979,20(7):1441-1443
13. U.Zimmermann, et al., Electric field-induced cell to cell fusion, J.Memb.Biol., 1982, 67(3):165-182
14. U. Zimmermann, et al., Electric field-induced release of chloroplasts from plant protoplasts,Naturwissenschaften, 1982,69:451-452
15. M. Nishiguchi, et al., An improved method for electroporation in plant protoplasts:
infection of tobacco protoplasts by tobacco mosaic virus particles, Plant Cell Reports,1987, 6(2):90-93
16. Donald C.Change, et al, Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy, Biophys J. 1990, 58(1): 1-12
17．张弘，刘长军，王保义等．低强度瞬态电磁脉冲引发细胞膜电穿孔的试验初探．细胞生物学杂志．1999，22(1)
18．汪和睦，李金慧．细胞电穿孔导入DNA的动态特性．微生物学通报．1994，21(2)
19．刘缨，张锦珠．细胞电穿孔动态过程的荧光测量．生物物理学报．1994，10(2)
20．张锦珠，孔可健，脉冲电场对人红细胞膜结构影响的冷冻断裂研究，试验生物学报，1994，27(2)：183-191
21. R.Benz, U.Zimmermann, High electric field effects on the cell membranes of Halicystic parvula, Planta, 1981, 152(4):314-318
22. Dimiter S.Dimitrov, Rakesh K.Jain. Membrane Stability. Biochimica et Biophysica Acta. 1984,779:437-468
23. U.Zimmermann,Electric field-mediated fusion and related electrical phenolmena, Biochimica et Biophy.Acta,1982,694(3):227-277
24. Arthur E.Sowers, A long-lived fusogenic state is induced in Erythrocyte Ghosts by electric pulses, Journal of Cell Biol., 1986,102(4):1358-1362
25. W. Mehrle, et al.,Evidence for asymmetrical uptake of fluorescent dyes through electro-permeabilized membranes of avena mesophyll protoplasts. FEBS Letters, 1985,185-89-94
26. I.G. Abidor, Electric breakdown of bilayer lipid membranes: Ⅰ. The main experimental facts and their qualitative discussion,, 1979,6:37-52
27. Hermann Berg, et al., Possibilities of cell fusion and transformation by electrostimulation,Bioelectrochem.Bioenerget., 1984,12:119-133
28. Tadej Kotnik, Damjian Miklavi,Analytical Description of Transmembrane Voltage Induced by Electric Fields on Spheroidal Cells.Biophysical Journal,2000, 79:670-679
29. U.Zimmermann, G.Pilwat, et al., Effects of External Electrical Fields on Cell Membranes,Bioelectrochem.Bioenerg.,1976,3:58-83
30. Herzog, R., et al., Life Science, 1986, 39:2279-2288
31. Weaver J C. Electroporationin cells and tissues: A biophysical phenonmenon due to electromagmetic fields, Radio Science, 1995, 30(1): 205
32. Wong T K, Neumarme, Electric field mediated gene transferr, Biochem Biophys Res Commun, 1982,107:584-587
33. Karl H.Schoenbach, Sunao Katsuki, Robert H.Stark, E.Stephen Buescher, and Stephen J.Beebe, Bioelectrics-new applications for pulsed power technology[J], IEEE Trans on plasma science, 2002,30(1): 293-300.
34. Mouneimney, Electroinsertion of full length recombineant CD4 in to red blood cell membrane, Biochem. Biophys Acta,1990,1027:53-58
35. Mirlm, Electrochemotherapy: potentialtion of antitumore effect of bleomycin by local electric pulsed, Eur J Cancer, 1991,27:68-72
36．姚成果，孙才新．陡脉冲不可逆电击穿恶性肿瘤细胞的研究，高电压技术，2003，29(1)：45-47
37. Weaver JC.,Recent advances in skin electroporation: mechanism and efficacy.In:2nd World congress for EMBM Abstract Book, Bologna, Italy June 8-13,1994:44
38．张占松，蔡宣三等编著，开关电源的原理与设计，电子工业出版社，p1-3．
39．张芝涛等，脉宽调制型(PWM)高压电源的研究，电力环境保护，1994，10(1)：35-38
40．冯挹，30kHz高频开关电源变压器的设计，http://www.willar.com
41．张文利，高压大功率开关电源技术的研究(硕士论文)
42．李希茜，高频变压器的设计，现代电子技术，2001年第9期，p7-8
43．彭勇，王子淑等，低强度瞬态电磁场下动物细胞的电穿孔效应，四川大学学报，2000，37(4)：586-589
44．(美) F．奥斯伯等著，颜子颖，王海林译，精编分子生物学试验指南，北京，科学出版社，1998．6，pp793-800
45．王保义，杨杰斌等，毫微秒电磁脉冲的生物效应试验研究和机理分析，中国科学(C辑)，1997，27(1)：35-39
46．邹积岩，吴为民，脉冲电场食品处理技术，高电压技术，2000，26(6)：31-33
47．汪和睦，汪洲，细胞电穿孔及电融合的机理及应用，生物化学与生物物理进展，1994，21(4)：322-326
48．王保义，刘长军，电穿孔——解释生物体中非热生物效应的关键机理，四川大学学报，1997，34(3)：310-314
49. J.M.Crowley, Electrical breakdown of biomolecular lipid membranes as an electromechartical instability, Biophys.J., 1973,13:711-724
50．牛中奇，细胞膜上自电场应力，中国生物医学工程学报，1994，13(1)：90-95
51. D.S. DIMITROV, Electroporation and Electrofusion of Membranes,. Handbook of Biological Physics, Volume 1.edited by R. Lipowsky and E. Sackmann.
52. Barry H.Honig, Electrostatic interactions in membranes and proteins. Ann. Rev. Biophys. Chem., 1986,15:163-93
53. J.A.Stratton, Electromagnetic Theory, McGraw-Hill Book Company Inc., New York,NY,USA,1941.
54. I.Krakovsky, T.Romijn, et al.,A few remarks on the electrostriction of elastomers, Journal of applied physics, 1999,85(1):628-629
55. T.J.Lewis, Electromechanical effects in biological membrane,2002 Annual report conference on electrical insulation and dielectric phenomena, pp87-90
56. J.N.Israelachvili, S.Marcelja and R.G.Horn, Physical Principles of Membrane Organization, Quarterly Rev. of Biophys., 1980,13:121-200
57. J.N.Israelachvili, Intermolecular and Surface Forces,2nd Edition, Academic Press, London
58. Stephen J.Beebe, et al., Nanosencond Pulsed Electric Field(nsPEF) Effects on Cells and Tissues: Apoptosis Induction and Tumor Growth Inhibition, IEEE transactions on plasma science,2002, 30(1): 286-292
59. R.P.Joshi,Q.Hu and K.H.Schoenbach, Dynamical Modeling of Cellular Response to Short High-Intensity Electric Fields, Workshop on "Dielectric Properties and Electrical Breakdown of Biological Membranes", October 20,2002
60. Karl H.Schoenbach, The effect of pulsed electric fields on biological cells: experiments and applications,IEEE Trans on plasma science, 1997,25(2):284-292.
61. Karl H.Schoenbach, Stephen J.Beebe, and E.Stephen Buescher. Intracellular effect of
ultrashort electrical pulses[J], Bioelectromagnetics, 2001,22(6):440—448
62. Martin Garderse. Utrashort pulse electroporation: application of high pulsed electric fields to induced caspase activation of human lymphocytes[J], IEEE Trans on plasma science, 2000, 28(2): 667-670.
63. T.Kotnik, F.Bobanovic, D.Miklavacic, "Sensitivity of transmembrane voltage induced by applied electric fields-a theoretical analysis", Bioelectrochem. Bioenerg. 1997, 43: 285-291.
64. Tadej Kotnik, Damijan Miklavi. Theoretical Evaluation of the Distributed Power Dissipation in Biological Cells Exposed to Electric Fields[J]. Bioelectromagnetics, 2000,21:385-394
65. Kenneth R. Foster. Thermal and nonthermal mechanisms of interaction of radio-frequency energy with biological systems, IEEE Trans on plasma science, 2000, 28(1): 15-23