电磁脉冲诱导细胞膜电穿孔及其在治疗肿瘤中的应用
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摘要
细胞膜是细胞外大分子进入细胞内的主要屏障，电穿孔(Electroporation)现象是指在电磁脉冲(Electromagnetic Pulses，EMP)作用下，细胞膜两边产生感应电位，破坏了细胞膜的绝缘性，从而使细胞膜的稳定性降低，瞬时增强细胞膜的通透性，在细胞膜的双脂层上形成瞬时可逆微孔的生物物理现象。电穿孔现象创造了一条通向细胞内的通道，可促进抗癌药物等大分子进入细胞内部，这种利用电穿孔现象将抗癌药物导入细胞内的治疗肿瘤的方法被称为肿瘤的电穿孔治疗。
在本论文中，我们叙述了细胞膜电穿孔的过程，通过实验的方法观察到了HeLa等细胞在电磁脉冲作用下的电穿孔现象，从实验总结获得了细胞膜电穿孔的最佳电磁脉冲参数；我们研究并报道了电磁脉冲对治疗肿瘤的作用，在实验装置、实验方法等方面都进行了有益的探索，并取得了良好的实验结果，研究结果得到同行专家关注与认可，实验结果处于国内研究的前列。为进一步的动物实验研究以及临床肿瘤治疗的研究提供了可供参考的方法和数据。我们发现：通过小动物实验，在电磁脉冲和抗癌药物的共同作用下，接种在小动物身上的肿瘤能够得到有效的抑制或治愈，肿瘤的完全响应率达到65％，部分响应率为10％，可见治疗效果是很明显的。本论文分为四个部分，第一部分为在电磁脉冲作用下细胞的电穿孔效应、原理和机制，第二部分叙述了电磁脉冲导致细胞膜电穿孔的实验研究，第三部分研究了电磁脉冲在治疗肿瘤中作用，包括实验过程和基本结果。第四部分总结了电穿孔治疗的现状及其展望。
Cell membranes are the mainly barrier for the exchange of molecules between the ceil interior and exterior. When cells are subjected to the electromagnetic pulse (EMP), the membrane permeability barrier is temporarily suppressed and the membrane stability is decreased, this was termed electroporation. Electroporation is the physical process of inducing transient permeability of cell membranes by EMP, so Electroporation provide a physical method for entering the cell interior, and increase the anti-cancer drugs into the cell interior greatly.
In this paper, we reported that the process of electroporation, and observed the electroporation of HeLa by experiment, and got the perfect parameters of EMP for the electroporation; on the other hand, we reported a new approach for the treatment of sarcoma-Electroporation therapy (EPT). Electroporation can accelerate molecules like anticancer drugs into cytoplasm by electromagnetic pulses (EMP). The EPT are used to treat the S180 sarcomas of the KunMing mice by intratumoral injection of low dosage of drugs. From the experiment, we discovered that this approach could obtain remarkable effect The paper is divided into four parts, in the first part, we described the principles of electroporation; in the second part, we described the electroporation of cells induced by the EMPs; in the third part, we studied the method of EPT, and got some results; the fourth part is the summary and outlook.
在本论文中，我们叙述了细胞膜电穿孔的过程，通过实验的方法观察到了HeLa等细胞在电磁脉冲作用下的电穿孔现象，从实验总结获得了细胞膜电穿孔的最佳电磁脉冲参数；我们研究并报道了电磁脉冲对治疗肿瘤的作用，在实验装置、实验方法等方面都进行了有益的探索，并取得了良好的实验结果，研究结果得到同行专家关注与认可，实验结果处于国内研究的前列。为进一步的动物实验研究以及临床肿瘤治疗的研究提供了可供参考的方法和数据。我们发现：通过小动物实验，在电磁脉冲和抗癌药物的共同作用下，接种在小动物身上的肿瘤能够得到有效的抑制或治愈，肿瘤的完全响应率达到65％，部分响应率为10％，可见治疗效果是很明显的。本论文分为四个部分，第一部分为在电磁脉冲作用下细胞的电穿孔效应、原理和机制，第二部分叙述了电磁脉冲导致细胞膜电穿孔的实验研究，第三部分研究了电磁脉冲在治疗肿瘤中作用，包括实验过程和基本结果。第四部分总结了电穿孔治疗的现状及其展望。
Cell membranes are the mainly barrier for the exchange of molecules between the ceil interior and exterior. When cells are subjected to the electromagnetic pulse (EMP), the membrane permeability barrier is temporarily suppressed and the membrane stability is decreased, this was termed electroporation. Electroporation is the physical process of inducing transient permeability of cell membranes by EMP, so Electroporation provide a physical method for entering the cell interior, and increase the anti-cancer drugs into the cell interior greatly.
In this paper, we reported that the process of electroporation, and observed the electroporation of HeLa by experiment, and got the perfect parameters of EMP for the electroporation; on the other hand, we reported a new approach for the treatment of sarcoma-Electroporation therapy (EPT). Electroporation can accelerate molecules like anticancer drugs into cytoplasm by electromagnetic pulses (EMP). The EPT are used to treat the S180 sarcomas of the KunMing mice by intratumoral injection of low dosage of drugs. From the experiment, we discovered that this approach could obtain remarkable effect The paper is divided into four parts, in the first part, we described the principles of electroporation; in the second part, we described the electroporation of cells induced by the EMPs; in the third part, we studied the method of EPT, and got some results; the fourth part is the summary and outlook.
引文
1．李缉熙，牛中奇。生物电磁学概论[M]。第一版，西安电子科技大学出版社，1990年。
2．高攸纲，刁庆安等。电磁环境对生态的危害及防护。基础医学与临床。2000．20(1)
3．王保义，唐敬贤，江汉保，等。电磁场在生物医学中应用[M]。第一版，北京：国防工业出版社，1990年。
4．刘亚宁。电磁辐射生物效应的机理研究述评。基础医学与临床。2000．20(1)
5．王保义，杨杰斌，等。毫微秒电磁脉冲的生物效应实验研究和机理分析。中国科学(C辑)，1996年10月，第27卷第1期。
6．王保义，刘长军，等。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(自然科学版)。1997年6月，第34卷，第3期。
7. B. Y. Wang C. J. Liu H. Zhang, Membrane electroporation due to weak transient electromagnetic pulses. The 4th Asia-Pacific Conference on medical and Biomedical Engineering.
8．张弘，王保义，刘长军，等。利用弱电磁脉冲对细胞电穿孔提高抗癌药物疗效的研究。电波科学学报。1999年4月。
9．刘长军，张弘，王保义，等。瞬态电磁脉冲对细胞跨膜电位的影响。四川大学学报(自然科学版)。1999年6月，第36卷，第5期。
10．刘长军，王保义，张弘，等。低强度快速电磁脉冲导致细胞电穿孔的研究。科学通报，1999年6月，第44卷，第11期。
11．刘长军，王保义，张弘，等。脉冲电磁场对细胞模型特异性作用机理研究。生物医学工程杂志，1998年，15(3)。
12. C. J. Liu B. Y. Wang H. Zhang, Animal erythrocytes membrane deformation due to low-intensity transient electromagnetic pulses. The second international EMF seminar.
13. Changjun Liu, Baoyi Wang, et al, Cell Deformation and Increase of Cytotoxicity of Anticancer Drugs Due to Low-Intensity Transient
Electromagnetic Pulses. IEEE Transactions on Plasma Science. Vol. 28. No. 1, February 2000
14．王保义，张弘，刘长军，等。利用电磁辐射引起细胞膜电穿孔进行癌症治疗的研究。电磁辐射生物效应及其医学应用。2001年9月。
15. B. Y. Wang G. Yang H. Zhang, The study of enhancing the cytotoxicity of antitumor drugs through EMP. 2002 3rd international conference on microwave and millimeter wave technology proceedings.
16. B. Y. Wang H. C. Chen H. Zhang, The treatment of sarcomas by the electroporation therapy. 2002 3rd international conference on microwave and millimeter wave technology proceedings.
17．邹方东，王子淑，王保义等。瞬态电磁场(TEMF)对细胞膜的作用。四川大学学报(自然科学版)1998年12月，第35卷，论文集。
18．熊兰，孙才新，廖瑞金等。细胞膜电穿孔的机理及应用前景的初步探讨。重庆大学学报(自然科学版)2000年7月，第23卷，第4期。
19. Schwan H. P. Electro and Magnetbiology. 1994. 13(2)
20．田心棣。人造双分子膜。高等教育出版社。1987
21. Katherine A. Debruin, Wanda Krassowska et al. Modeling Electroporation in a single cell. I. Effects of Field Strength and Rest Potential. Biophysical Journal. Vol. 77. September 1999
22. Gailey P C, Easterly C E. Cell membrane potentials induced during exposure to EMP fields. Electro-and Magnetobiology, 1994, 13(2): 159-165
23. Tsong T. Y. Bioelectrochem. Bioenergy. 1990, 24(3)
24. Chang D. C. Reese T. S. Changs in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys. J. 1990: 58(1)
25. Sukbendu B. Dev, D. P. Rabussay, et al. Medical Applications of Electroporation IEEE Transactions on Plasma Science. Vol. 28. No. 1. February 2000
26. E. Newmann, et al., EMBO, 1(7), 1992
27. Mouneimne Y. Electroinsertion of full length recombinant cd4 into red blood cell membrane. Biochem., Biophys., Acta, 1990
28. Tsong T. Y. Electroporation of membrane. Biophys. J. 60, 1991
29. Dower W J. Cwirla S E.'Guide to electoporation and electrofusion. San Dieago, Acaden Press, 1992
30. Sale A. JH, Hamilton W. A, Effects of electric fields on micro-organizm killing of bacteria and yeasts. Biochim. Biophys Acta, 1967
31. Zimmermann. U and J. Vienken. Electric field-induced cell fusion. J. Membrane Biol. 1982, 67: 165-182
32. Neumann E, Rosenheck K. Permeability changes included by electrical pulse in vesicular membranes. J Membr. Biol. 1982
33．汪和睦，李金慧。细胞电穿孔导入DNA的动态特征。微生物学通报。1994年21(2)
34. Zimmermann. U et al. Biophys. 1994, 14: 881-899
35. K inosita K. J, Tsong T. Y. Voltage includes formation and hemolysis of human erythocytes. Biochim. Biophys. Acta, 1979.
36. Hofmann G. A, Evans G. Electronic genetic-physical and biological aspects of cellular electro manipulation. IEEE Eng. Med. Biol. Mag. 1986, 5(4)
37. Weaver J. C, Electroporation: A dramatic, nonthermal electric field phenomenon. In: Proceedings of the first world congress for electricity and magnetism in biology and medicine. Lake Buena Vista, Florida: Academic Press, 1992.
38．罗深秋，医用细胞生物学。军事医学科学出版社，1998年
39. Gunter A. Hofmann, Sukhendu B Dev. Electrochemotherapy: Transition from laboratory to the clinic. IEEE engineering in medicine and biology. November/December, 1996.
40. G. A. Hofmann, S. B. Dev, et al. Electroporation therapy: A new approach for the treatment of head and neck cancer. IEEE transactions on biomedical engineering. Vol. 46, No. 6, June 1999
41. Heller R. J. et al. Phase Ⅰ /Ⅱ trial for the treatment of cutaneous and subcutaneous tumors using electrotherapy. Cancer, 1996, Vol. 77
42. Damijan Miklavcic, et al. The importance of electric field distribution for effective in vivo electroporation of tissues. Biophysical Journal. Vol. 74, May 1998
43．许宝强，袁其平等。基因脉冲导入仪的研制。天津工业大学学报。第21卷，第2期，2001年
44. Wang BY, Yang JB, Guo QG Experimental and mechanism analysis on bioeffects by nanosecond electromagnetic pulse. Science in China(C)1997
45．杨孔等。电磁脉冲各参数对Hela细胞可逆电穿孔影响的研究。四川大学学报，2001年8月，第38卷，第四期
46．孙敬儒等。电穿孔诱发细胞凋亡和提高抗癌药物细胞毒性的研究。四川大学学报，2002年4月，第39卷
47．李茂国译。细胞生存与死亡的社会性控制[J]。细胞生物学杂志，1993，15。
48．孙骥远，郑民华等，电穿孔化疗治疗结直肠癌的研究现状，国外医学，2001年，第21卷，第四期
49. M. Okino andH.Mohri, Effect of a high-voltage electrical impulse and an anticancer drug on in vivo growing tumors, Jpn. J. Cancer Res., Vol.78, 1987
50. Heller R. J. Bioelectrochem. Bioenerg. 1995, 36: 83
2．高攸纲，刁庆安等。电磁环境对生态的危害及防护。基础医学与临床。2000．20(1)
3．王保义，唐敬贤，江汉保，等。电磁场在生物医学中应用[M]。第一版，北京：国防工业出版社，1990年。
4．刘亚宁。电磁辐射生物效应的机理研究述评。基础医学与临床。2000．20(1)
5．王保义，杨杰斌，等。毫微秒电磁脉冲的生物效应实验研究和机理分析。中国科学(C辑)，1996年10月，第27卷第1期。
6．王保义，刘长军，等。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(自然科学版)。1997年6月，第34卷，第3期。
7. B. Y. Wang C. J. Liu H. Zhang, Membrane electroporation due to weak transient electromagnetic pulses. The 4th Asia-Pacific Conference on medical and Biomedical Engineering.
8．张弘，王保义，刘长军，等。利用弱电磁脉冲对细胞电穿孔提高抗癌药物疗效的研究。电波科学学报。1999年4月。
9．刘长军，张弘，王保义，等。瞬态电磁脉冲对细胞跨膜电位的影响。四川大学学报(自然科学版)。1999年6月，第36卷，第5期。
10．刘长军，王保义，张弘，等。低强度快速电磁脉冲导致细胞电穿孔的研究。科学通报，1999年6月，第44卷，第11期。
11．刘长军，王保义，张弘，等。脉冲电磁场对细胞模型特异性作用机理研究。生物医学工程杂志，1998年，15(3)。
12. C. J. Liu B. Y. Wang H. Zhang, Animal erythrocytes membrane deformation due to low-intensity transient electromagnetic pulses. The second international EMF seminar.
13. Changjun Liu, Baoyi Wang, et al, Cell Deformation and Increase of Cytotoxicity of Anticancer Drugs Due to Low-Intensity Transient
Electromagnetic Pulses. IEEE Transactions on Plasma Science. Vol. 28. No. 1, February 2000
14．王保义，张弘，刘长军，等。利用电磁辐射引起细胞膜电穿孔进行癌症治疗的研究。电磁辐射生物效应及其医学应用。2001年9月。
15. B. Y. Wang G. Yang H. Zhang, The study of enhancing the cytotoxicity of antitumor drugs through EMP. 2002 3rd international conference on microwave and millimeter wave technology proceedings.
16. B. Y. Wang H. C. Chen H. Zhang, The treatment of sarcomas by the electroporation therapy. 2002 3rd international conference on microwave and millimeter wave technology proceedings.
17．邹方东，王子淑，王保义等。瞬态电磁场(TEMF)对细胞膜的作用。四川大学学报(自然科学版)1998年12月，第35卷，论文集。
18．熊兰，孙才新，廖瑞金等。细胞膜电穿孔的机理及应用前景的初步探讨。重庆大学学报(自然科学版)2000年7月，第23卷，第4期。
19. Schwan H. P. Electro and Magnetbiology. 1994. 13(2)
20．田心棣。人造双分子膜。高等教育出版社。1987
21. Katherine A. Debruin, Wanda Krassowska et al. Modeling Electroporation in a single cell. I. Effects of Field Strength and Rest Potential. Biophysical Journal. Vol. 77. September 1999
22. Gailey P C, Easterly C E. Cell membrane potentials induced during exposure to EMP fields. Electro-and Magnetobiology, 1994, 13(2): 159-165
23. Tsong T. Y. Bioelectrochem. Bioenergy. 1990, 24(3)
24. Chang D. C. Reese T. S. Changs in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys. J. 1990: 58(1)
25. Sukbendu B. Dev, D. P. Rabussay, et al. Medical Applications of Electroporation IEEE Transactions on Plasma Science. Vol. 28. No. 1. February 2000
26. E. Newmann, et al., EMBO, 1(7), 1992
27. Mouneimne Y. Electroinsertion of full length recombinant cd4 into red blood cell membrane. Biochem., Biophys., Acta, 1990
28. Tsong T. Y. Electroporation of membrane. Biophys. J. 60, 1991
29. Dower W J. Cwirla S E.'Guide to electoporation and electrofusion. San Dieago, Acaden Press, 1992
30. Sale A. JH, Hamilton W. A, Effects of electric fields on micro-organizm killing of bacteria and yeasts. Biochim. Biophys Acta, 1967
31. Zimmermann. U and J. Vienken. Electric field-induced cell fusion. J. Membrane Biol. 1982, 67: 165-182
32. Neumann E, Rosenheck K. Permeability changes included by electrical pulse in vesicular membranes. J Membr. Biol. 1982
33．汪和睦，李金慧。细胞电穿孔导入DNA的动态特征。微生物学通报。1994年21(2)
34. Zimmermann. U et al. Biophys. 1994, 14: 881-899
35. K inosita K. J, Tsong T. Y. Voltage includes formation and hemolysis of human erythocytes. Biochim. Biophys. Acta, 1979.
36. Hofmann G. A, Evans G. Electronic genetic-physical and biological aspects of cellular electro manipulation. IEEE Eng. Med. Biol. Mag. 1986, 5(4)
37. Weaver J. C, Electroporation: A dramatic, nonthermal electric field phenomenon. In: Proceedings of the first world congress for electricity and magnetism in biology and medicine. Lake Buena Vista, Florida: Academic Press, 1992.
38．罗深秋，医用细胞生物学。军事医学科学出版社，1998年
39. Gunter A. Hofmann, Sukhendu B Dev. Electrochemotherapy: Transition from laboratory to the clinic. IEEE engineering in medicine and biology. November/December, 1996.
40. G. A. Hofmann, S. B. Dev, et al. Electroporation therapy: A new approach for the treatment of head and neck cancer. IEEE transactions on biomedical engineering. Vol. 46, No. 6, June 1999
41. Heller R. J. et al. Phase Ⅰ /Ⅱ trial for the treatment of cutaneous and subcutaneous tumors using electrotherapy. Cancer, 1996, Vol. 77
42. Damijan Miklavcic, et al. The importance of electric field distribution for effective in vivo electroporation of tissues. Biophysical Journal. Vol. 74, May 1998
43．许宝强，袁其平等。基因脉冲导入仪的研制。天津工业大学学报。第21卷，第2期，2001年
44. Wang BY, Yang JB, Guo QG Experimental and mechanism analysis on bioeffects by nanosecond electromagnetic pulse. Science in China(C)1997
45．杨孔等。电磁脉冲各参数对Hela细胞可逆电穿孔影响的研究。四川大学学报，2001年8月，第38卷，第四期
46．孙敬儒等。电穿孔诱发细胞凋亡和提高抗癌药物细胞毒性的研究。四川大学学报，2002年4月，第39卷
47．李茂国译。细胞生存与死亡的社会性控制[J]。细胞生物学杂志，1993，15。
48．孙骥远，郑民华等，电穿孔化疗治疗结直肠癌的研究现状，国外医学，2001年，第21卷，第四期
49. M. Okino andH.Mohri, Effect of a high-voltage electrical impulse and an anticancer drug on in vivo growing tumors, Jpn. J. Cancer Res., Vol.78, 1987
50. Heller R. J. Bioelectrochem. Bioenerg. 1995, 36: 83