陡脉冲在活体兔肝组织的电场分布和剂量研究
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摘要
陡脉冲疗法是在电脉冲化学疗法的基础上发展起来的一种新的局部物理治疗肿瘤技术,它是在保证患者安全的前提下,将适当参数组合的脉冲引入肿瘤组织,使肿瘤细胞发生不可逆性电击穿,导致肿瘤细胞死亡。前期已有细胞、离体肿瘤组织和动物实验的大量研究结果证实陡脉冲疗法对肿瘤的有效杀伤和抑制作用,但有关陡脉冲在活体组织中的电场分布和治疗剂量学研究尚少。
目的本课题主要研究陡脉冲作用于活体兔肝组织的电场分布;探讨陡脉冲电场与杀伤效应的量效关系;探索陡脉冲电场致新西兰兔肝细胞发生可逆性和不可逆性电穿孔的阈值。
材料与方法以正常新西兰兔肝组织为实验对象,将不同剂量的陡脉冲施加于新西兰兔肝组织,72 h后处死取材,TTC染色和HE染色观察其坏死形态,描绘其坏死轮廓;将不同剂量的陡脉冲作用于48只清洁级新西兰兔肝组织,测量每一剂量下兔肝组织的坏死面积;将固定脉宽10 us、频率20 Hz和时间10 min,梯度增加电压的陡脉冲电场作用于兔肝组织,以博莱霉素为标记物,通过TTC染色、HE染色和电镜观察肝细胞的损伤效应。
结果不同剂量的陡脉冲作用于活体兔肝组织后形成的坏死形态不同。随着陡脉冲剂量的增加,坏死形态由以每个电极针为中心的圆形或椭圆形独立的坏死灶发展到12个电极针包围范围内长方形坏死灶;陡脉冲致兔肝组织的坏死面积随着陡脉冲剂量的增大而增加;固定脉宽10 us、频率20 Hz和时间10 min,当电压为750 V时,细胞核出现染色质浓缩;当电压为800 V时,细胞出现凝固性坏死。
结论陡脉冲在活体兔肝组织中的电场分布与理论三维电场仿真分布相似;坏死面积与陡脉冲剂量有明显的量效关系,其值随着剂量的增大而增加;固定脉宽10 us、频率20 Hz和时间10 min,当电压为750 V时,肝细胞发生可逆性电击穿,即肝细胞可逆性电穿孔的电压阈值为750 V;当电压为800 V时,肝细胞发生不可逆性电击穿,即肝细胞不可逆性电穿孔的电压阈值为800 V。
Steep pulse therapy is a new local physical approach in the treatment of neoplasm, which is based on electrical impulse chemotherapy. suitable steep pulsed electrical fields is delivered to the tumor, and causes irreversible cell damage. the massive cells, the exsomatize tumor and the animal experiments have confirmed the steep pulse therapy can kill the tumor effectively. But steep pulsed electric fields distribution in organization and the treatment dosiology research has not yet been reported systemly. In our experiment the liver of rabbits in vivo was exposed to steep pulsed electrical fields with serial dosage to discuss its electric fields distribution in the liver, and to get quantity-effect relations between the electrical field therapeutic dosage and the casualty effect.
Object The aim of the present study was to observe steep pulsed electric fields distribution on liver of rabbits in vivo, to explore quantity-effect relations between the electric field therapeutic dosage and the casualty effect, and to analysis the steep pulsed electric field intensities about the irreversible permeabilisation threshold and reversible permeabilisation threshold in the liver of rabbits.
Material and method the liver of rabbits in vivo was exposed to steep pulsed electrical fields with serial dosage, the rabbits were sacrificed after 72 h, TTC and histological examination were observed the change of morphology and pathology in the liver exposed and describe necrosis contour; The liver of 48 rabbits in vivo was exposed to steep pulsed electrical fields with serial dosage, the rabbits were sacrificed after 72 h, and necrosis area was surveyed; When the liver of rabbits in vivo was exposed to steep pulsed electrical fields with voltage of serial dosage which was fixed pulse width of 10 us, the frequency of 20 Hz and the time of 10 min, histological examination and electron microscope examination were used to observe the change of morphology and pathology in the liver marked the electroporation with bleomycin.
Results The liver of rabbits exposed to steep pulsed electric fields of serial dosage resulted in the different necrosis contour which varied from the central circular or ellipse about each electrode to square shape surrounding 12 electrode needles; When the liver of rabbits in vivo was exposed to electrical pulsed fields of serial dosage, liver necrosis area increased gradually with the increasing of the electric-field intensity; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 750 V voltage which was fixed pulse width of 10 us, the frequency of 20 Hz and the time of 10 min, the liver cell nucleus appeared the chromatin concentration. When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 800 V voltage which was fixed pulse width of 10 us, the frequency of 20 Hz and the time of 10 min , the liver cell were to observed to be coagulation necrosis.
Conclusion steep pulsed electric fields distribution on liver of rabbits in vivo was similar with the three dimensional emulate drawing of electric fields distribution; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of serial dosage , liver necrosis area increased with the increasing of steep pulse dosage, and we got quantity-effect relations between the electric field therapeutic dosage and the casualty effect; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 750 V voltage which is fixed with the pulse width of 10us and the frequency of 100 Hz, the liver cell nucleus appeared the chromatin concentration, So we considered that the steep pulsed electric fields of 750V voltage could be the reversible permeabilisation threshold of liver cell in vivo; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 800 V voltage which is fixed with the pulse width of 10 us and the frequency of 100 Hz, the liver cell were to observed to be coagulation necrosis, So we considered that the steep pulsed electric fields of 750V voltage could be the reversible permeabilisation threshold of rabbit liver cell in vivo.
目的本课题主要研究陡脉冲作用于活体兔肝组织的电场分布;探讨陡脉冲电场与杀伤效应的量效关系;探索陡脉冲电场致新西兰兔肝细胞发生可逆性和不可逆性电穿孔的阈值。
材料与方法以正常新西兰兔肝组织为实验对象,将不同剂量的陡脉冲施加于新西兰兔肝组织,72 h后处死取材,TTC染色和HE染色观察其坏死形态,描绘其坏死轮廓;将不同剂量的陡脉冲作用于48只清洁级新西兰兔肝组织,测量每一剂量下兔肝组织的坏死面积;将固定脉宽10 us、频率20 Hz和时间10 min,梯度增加电压的陡脉冲电场作用于兔肝组织,以博莱霉素为标记物,通过TTC染色、HE染色和电镜观察肝细胞的损伤效应。
结果不同剂量的陡脉冲作用于活体兔肝组织后形成的坏死形态不同。随着陡脉冲剂量的增加,坏死形态由以每个电极针为中心的圆形或椭圆形独立的坏死灶发展到12个电极针包围范围内长方形坏死灶;陡脉冲致兔肝组织的坏死面积随着陡脉冲剂量的增大而增加;固定脉宽10 us、频率20 Hz和时间10 min,当电压为750 V时,细胞核出现染色质浓缩;当电压为800 V时,细胞出现凝固性坏死。
结论陡脉冲在活体兔肝组织中的电场分布与理论三维电场仿真分布相似;坏死面积与陡脉冲剂量有明显的量效关系,其值随着剂量的增大而增加;固定脉宽10 us、频率20 Hz和时间10 min,当电压为750 V时,肝细胞发生可逆性电击穿,即肝细胞可逆性电穿孔的电压阈值为750 V;当电压为800 V时,肝细胞发生不可逆性电击穿,即肝细胞不可逆性电穿孔的电压阈值为800 V。
Steep pulse therapy is a new local physical approach in the treatment of neoplasm, which is based on electrical impulse chemotherapy. suitable steep pulsed electrical fields is delivered to the tumor, and causes irreversible cell damage. the massive cells, the exsomatize tumor and the animal experiments have confirmed the steep pulse therapy can kill the tumor effectively. But steep pulsed electric fields distribution in organization and the treatment dosiology research has not yet been reported systemly. In our experiment the liver of rabbits in vivo was exposed to steep pulsed electrical fields with serial dosage to discuss its electric fields distribution in the liver, and to get quantity-effect relations between the electrical field therapeutic dosage and the casualty effect.
Object The aim of the present study was to observe steep pulsed electric fields distribution on liver of rabbits in vivo, to explore quantity-effect relations between the electric field therapeutic dosage and the casualty effect, and to analysis the steep pulsed electric field intensities about the irreversible permeabilisation threshold and reversible permeabilisation threshold in the liver of rabbits.
Material and method the liver of rabbits in vivo was exposed to steep pulsed electrical fields with serial dosage, the rabbits were sacrificed after 72 h, TTC and histological examination were observed the change of morphology and pathology in the liver exposed and describe necrosis contour; The liver of 48 rabbits in vivo was exposed to steep pulsed electrical fields with serial dosage, the rabbits were sacrificed after 72 h, and necrosis area was surveyed; When the liver of rabbits in vivo was exposed to steep pulsed electrical fields with voltage of serial dosage which was fixed pulse width of 10 us, the frequency of 20 Hz and the time of 10 min, histological examination and electron microscope examination were used to observe the change of morphology and pathology in the liver marked the electroporation with bleomycin.
Results The liver of rabbits exposed to steep pulsed electric fields of serial dosage resulted in the different necrosis contour which varied from the central circular or ellipse about each electrode to square shape surrounding 12 electrode needles; When the liver of rabbits in vivo was exposed to electrical pulsed fields of serial dosage, liver necrosis area increased gradually with the increasing of the electric-field intensity; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 750 V voltage which was fixed pulse width of 10 us, the frequency of 20 Hz and the time of 10 min, the liver cell nucleus appeared the chromatin concentration. When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 800 V voltage which was fixed pulse width of 10 us, the frequency of 20 Hz and the time of 10 min , the liver cell were to observed to be coagulation necrosis.
Conclusion steep pulsed electric fields distribution on liver of rabbits in vivo was similar with the three dimensional emulate drawing of electric fields distribution; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of serial dosage , liver necrosis area increased with the increasing of steep pulse dosage, and we got quantity-effect relations between the electric field therapeutic dosage and the casualty effect; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 750 V voltage which is fixed with the pulse width of 10us and the frequency of 100 Hz, the liver cell nucleus appeared the chromatin concentration, So we considered that the steep pulsed electric fields of 750V voltage could be the reversible permeabilisation threshold of liver cell in vivo; When the liver of rabbits in vivo was exposed to steep pulsed electric fields of 800 V voltage which is fixed with the pulse width of 10 us and the frequency of 100 Hz, the liver cell were to observed to be coagulation necrosis, So we considered that the steep pulsed electric fields of 750V voltage could be the reversible permeabilisation threshold of rabbit liver cell in vivo.
引文
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[4] Hofmann G. A, Dev S.B, Dimmer S, et al. Electroperation therapy: a new approach for the treatment of head and neck cancer[J]. IEEE Trans on Biomedical Engineering.1999, (46)6: 752~759.
[5] Stephen J. Beebe, P. M. Fox, L. J. Rec, et al. Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition[J]. IEEE Trans. on Plasma Science.2002, 30(1): 286~292.
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[2] Samuel Baron, Joyce Poast, Danielle Rizzo,et al. Electroporation of antibodies, DNA, and other macromolecules into cells: a highly efficient method[J]. Journal of Immunological Methods. 2000, (242): 115～126.
[3] Hofmann G. A, Evans G, et al.Electronic genetic-physical and biological aspects of cellular electromanipulation[J]. IEEE Eng. Med. Biol. Mag.1986, 5(4): 6～25.
[4] Hofmann G. A, Dev S.B, Dimmer S, et al. Electroperation therapy: a new approach for the treatment of head and neck cancer[J]. IEEE Trans on Biomedical Engineering.1999, (46)6: 752~759.
[5] Stephen J. Beebe, P. M. Fox, L. J. Rec, et al. Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition[J]. IEEE Trans. on Plasma Science.2002, 30(1): 286~292.
[6] Stephen J. Beebe,Paula M. Fox, Laura J.Rec, et al. Nanosecond,high-intensity pulsed electric fields induce apoptosis in human cells[J].FASEB J. 2003, 17(11):1493～1495.
[7] Ulrich Zimmermann, Uwe Friedrich, Heiko Mussauer, et al. Electromanipulation of mammalian cells: fundamentals and application[J]. IEEE Transaction on Plasma Science. 2000,28(1): 72~82.
[8] Sukhendu B. Dev, Dietmar P. Rabussary, Georg Widera,et al. Medical Applications of Electroporation[J]. IEEE Transaction on Plasma Science. 2000. 28(1): 206~223
[9] M. M. Kekez, P. Savic, B. F. Johnson,et al. Contribution to the biophysics of lethal effects of electric field on microorganisms[J]. Biochimica et Biophysica Acta. 1996, 79~88.
[10] K. H. Schoenbach, Amr. Abou-Ghazala, Terry. Vithoulkas, et al. Effect of pulsed electrical fields on biological cells[J]. Digest of Technical Papers-IEEE International Pulsed Power Conference. 1997, 29(2):73~78.
[11] 姚陈果,孙才新,米 彦,等.陡脉冲不可逆性电击穿恶性肿瘤细胞的研究[J],高电压技术.2003, 29,(1):45~47.
[12] Davalos RV, Mir IL, Rubinsky B.Tissue ablation with irreversible electroporation. [J]Ann Biomed Eng.2005, 33(2):223~231.
[13] Xiong lan ,Sun Caixin,Li Daqiang,et al.The experimental research of the effect of energy-controllable steep pulse on tumor tissue in vitro[J].Biomed Eng .2002, 19 (3):440~443.
[14] 李大强,熊兰,胡丽娜,等.能量可控陡脉冲对新西兰大白兔肝脏及肾脏组织损伤效应的初步探讨[J].中华实用医学,2003, l.5(3):1~4.
[15] 李均.陡脉冲电场对实验兔 VX2 乳腺移植瘤毛细淋巴管杀伤效应的实验研究[D].重庆医科大学.2005,6.
[16] 喻金梅,熊正爱,凌晓娟,等.陡脉冲对离体牛肝损伤效应的剂量学初探[J].第三军医大学学报.2006,28(24):2419~2421.
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