高压纳秒脉冲电场对同源细胞的杀伤差异性研究

doi:10.19595/j.cnki.1000-6753.tces.231473

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摘要

为深入研究高压纳秒脉冲电场诱导同源细胞的生物电学效应的差异性,本文以人永生化角质形成细胞(HaCaT)、人黑色素瘤细胞(A375)和人黑色素瘤耐药细胞(A375/R)为研究对象,开展细胞悬液杀伤实验,并搭建肿瘤三维水凝胶模拟组织消融实验平台,分析高压纳秒脉冲电场对悬浮细胞的杀伤效应以及对模拟组织的消融效果。同时,结合单细胞真实几何尺寸建立有限元仿真模型,分析高压纳秒脉冲电场作用下几何尺寸对细胞核膜上穿孔面积以及穿孔数量的影响。实验结果显示,在高压纳秒脉冲作用下,正常细胞的存活率最高及消融面积最小,肿瘤耐药细胞的存活率最低及消融面积最大。实验与仿真结果表明,正常细胞、肿瘤细胞及其肿瘤耐药细胞对高压纳秒脉冲电场的敏感性存在显著差异,高压纳秒脉冲电场对正常细胞的效果最弱,对肿瘤耐药细胞的效果最强。研究表明高压纳秒脉冲电场在肿瘤消融领域具有不影响周边正常组织的潜在的临床应用价值。

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	彭文成
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	姚陈果

关键词 ：纳秒脉冲电场, 同源细胞, 细胞存活率, 水凝胶模拟组织, 杀伤差异性

Abstract：

In terms of biological effects caused by the pulsed electric fields, the duration of the nanosecond pulse is less than the charging time constant of the cell membrane (~1μs), which has more profound effects on the intracellular organelles, known as the intracellular electron-manipulation. Based on this, the nanosecond pulsed electric fields (nsPEFs) could have unique bioelectromagnetic properties in the field of tumor ablation. Therefore, we studied the killing effects of homologous cells under the action of nsPEFs to investigate the difference between homologous cells to high-voltage nsPEFs.
The homologous cells, human immortal keratinocytes (HaCaT), human melanoma cells (A375), and human melanoma chemoresistant cells (A375/R), were chosen as research objects. The homemade pulse generator can output high-voltage nanosecond pulses with flexible and adjustable parameters. The selected pulse parameters were 5-15 kV/cm of field intensity, 300 ns of pulse width, 1 Hz of repetition frequency, and 80 of pulse number. Cell viability assay based on CCK-8 was used to study the survival rate of homologous cells after pulse treatment. The ablation effect of nanosecond pulses was further studied based on hydrogel-simulated tissue. The lethal threshold for a certain ablation area was calculated based on the ablation area. Furthermore, the electroporation response of the single cell to the nanosecond pulse is simulated by the finite element simulation model.
The survival rate of the three cells decreased with the increase of the electric field intensity of nsPEFs. The results of the cell viability assay showed that under the same parameters of nsPEFs, the survival rate of normal cells was the highest, followed by tumor cells, and tumor chemoresistant cells had the lowest survival rate. The results of the hydrogel simulated tissue ablation experiment showed that under the same pulse parameters, the ablation area of tumor chemoresistant cells was the largest, reaching 20.28±0.85 mm², while the ablation area of normal cells was the smallest, only 10.26±0.49 mm², which is significant difference. Meanwhile, the lethal threshold of normal cells and chemoresistant cells was calculated to be 7.15±0.65 kV/cm and 4.75±0.15 kV/cm, respectively, with a difference of approximately 2.4 kV/cm. These results indicated that there were significant differences in the sensitivity of normal cells, tumor cells, and tumor chemoresistant cells to the high-voltage nanosecond pulsed electric fields, and the nsPEFs had the least effect on normal cells and the strongest effect on tumor chemoresistant cells.
To analyze the potential reasons for the killing difference of cells by nsPEFs, the cell size and nucleus size of HaCaT, A375, and A375/R cells were measured and recorded by fluorescence microscopy. There was no significant difference in the cell size between HaCaT, A375, and A375/R, but there was a significant difference in the nucleus size. Based on the real cell geometry, the electroporation area and the pore number in the nuclear membrane were simulated. The results suggested that the difference in cell response to nsPEFs may be related to the nucleus size. Hence, tumor chemoresistant cells with the larger nucleus were more susceptible to nsPEFs, which indicated that high-voltage nanosecond pulsed electric fields have potential clinical application in the field of tumor ablation without affecting surrounding normal tissues.

Key words： Nanosecond pulsed electric fields homologous cells cell viability hydrogel scaffolds killing difference

PACS:	TM836
	Q64

通讯作者: 姚陈果,男,1975年生,教授,博士生导师,研究方向为脉冲功率技术及其生物医学应用。E-mail：yaochenguo@cqu.edu.cn

作者简介: 彭文成,男,1994年生,博士研究生,研究方向为脉冲功率技术及其生物医学应用。E-mail：pengwencheng@cqu.edu.cn

引用本文:

彭文成, 唐潇, 刘红梅, 董守龙, 姚陈果. 高压纳秒脉冲电场对同源细胞的杀伤差异性研究[J]. 电工技术学报, 0, (): 9043-43. Peng Wencheng, Tang Xiao, Liu Hongmei, Dong Shoulong, Yao Chenguo. Study on the Killing Difference of Homologous Cells by High-Voltage Nanosecond Pulsed Electric Fields. Transactions of China Electrotechnical Society, 0, (): 9043-43.

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