细胞骨架在ns脉冲诱导肿瘤细胞凋亡中的作用

为了研究细胞骨架在ns脉冲诱导肿瘤细胞凋亡中的作用,将脉冲电场参数组合(电场强度10kV/cm,脉宽500ns,脉冲30个,频率1Hz)作用于细胞骨架裂解的Hep-G2细胞。利用Annexin-V和碘化丙碇(propidium i-odide,PI)双染法检测细胞凋亡和坏死情况;采用荧光分光光度计观测线粒体跨膜电位和细胞膜穿孔的变化情况。试验发现:肌动蛋白裂解后脉冲处理组的细胞凋亡和坏死大大降低(与单独脉冲处理组相比检验水平P<0.05),早期细胞凋亡由(16.94±2.87)%下降到(4.77±1.87)%;晚期凋亡和坏死由(20.94±3.09)%下降到(14.38±2.60)%;脉冲处理组和肌动蛋白裂解后脉冲处理组的细胞线粒体跨膜电位都出现下降,但后者的线粒体跨膜电位却明显高于前者(P<0.05);单独脉冲处理组和细胞骨架缺失后脉冲处理组二者在相同时间点时其PI荧光强度无明显差异,且PI荧光随时间而变化的规律亦相似。上述结果表明:细胞骨架的缺失抑制了ns脉冲诱导细胞凋亡的线粒体通路,进而降低细胞凋亡水平,但对细胞膜的穿孔情况无明显影响。     

Effects of submicrosecond, high intensity pulsed electric fields on living cells - intracellular electromanipulation

Development of technology to produce nanosecond duration pulsed electric fields has allowed examination of the effects of ultrashort duration, high intensity electric fields on living cells. Theoretically, high intensity (MV/m) electric field applications with durations of less than one microsecond, when shortened toward nanoseconds, should increasingly affect intracellular rather than surface membranes of living cells. Experimentally, square-wave, 60 ns duration, high energy (36-53 kV/cm) pulses applied in trains of 1-10 pulses result in progressive increases in the numbers of permeabilized intracellular granules in a human eosinophil cell model-without large surface membrane effects. Electron micrographic examination of cells treated in this way demonstrates alteration of intracellular granule morphology consistent with permeabilization of granule membrane, i.e., intracellular electromanipulation. Continuous microscopic examination of individual living cells exposed to long or short duration pulsed electric field applications shows that permeabilization of surface membrane (median 5 minutes) with anodic preference (electroporation) and prompt cellular swelling follow a single, long duration (100 microsecond) pulse. In contrast, after a single short duration (60 ns) pulse, onset of surface membrane permeability is delayed (median 17 minutes), the increased permeability shows no anodic preference, and cellular swelling is absent suggesting that these effects are due to intracellular electromanipulation rather than direct effects on the surface membrane. Submicrosecond, intense pulsed electric fields applied to living cells achieve preferential effects on intracellular rather than surface membranes, potentially providing new approaches for selective/generalized cell or tissue ablation, growth stimulation and tissue remodeling.     

细胞内电处理用ns脉冲发生器研究进展

为了便于全面了解目前细胞内电处理实验研究的方法和具体实施方案,综述了国内外用于细胞内电处理实验研究的ns脉冲发生器。从电容器充放电和传输线理论2方面介绍了这些发生器产生ns级、高强脉冲电场的工作原理;分析了用于细胞内电处理的ns脉冲发生器性能特点、发生器的电路结构、元器件选择和技术指标等。实验结果表明,这些发生器产生的ns脉冲电场能诱导细胞内电处理效应。对今后的研究工作进行了讨论和展望。     

Pulse generators for pulsed electric field exposure of biological cells and tissues

This paper describes three pulse generators: a spark gap switched coaxial cable, a spark gap switched Blumlein, and a solid state modulator, developed for applying ultrashort electrical pulses to biological materials in culture. Research has shown that ultrashort pulsed electric fields can induce apoptosis in biological cells, and that pulses as short as 10 ns with field amplitude greater than 1 W/m cause membrane phospholipid rearrangement and activation of the effector enzymes of apoptosis. Pulses of very short duration use only tens of mJ per mL per pulse to induce apoptosis and other intracellular effects without causing thermal trauma. The pulse generators discussed here, each of a different topology, deliver ns pulsed electric fields (nsPEF) to cells in liquid suspension, and can be modified to drive electrodes for external, surgical, or endoscopic treatment of tissues in situ.     

纳秒脉冲电场诱导肿瘤细胞凋亡的内质网信号通路分析

ns脉冲电场在诱导肿瘤细胞凋亡方面展现出诱人的应用前景。由于ns脉冲电场诱导肿瘤细胞凋亡的机制尚未明确,严重阻碍了ns脉冲电场肿瘤治疗的临床进程。为进一步揭示其诱导凋亡的机制,将参数组合(电压幅值9kV,脉宽100ns,脉冲30个,频率1Hz)作用于人卵巢浆液性囊腺癌(SKOV3)细胞。首先检测了半胱氨酸天冬氨酸蛋白酶-12(cysteine aspartic acid specific protease-12,Caspase-12)蛋白的释放水平,其次用浓度分别为0、25、50、100μmol/L的钙螯合剂(BAPTA-AM)螯合细胞内的游离Ca2+,用Hoechst 33342荧光染色检测细胞凋亡率,最后采用蛋白质印迹(western blot)技术检测了Caspase-12蛋白释放的变化。试验结果表明:ns脉冲诱导SK-OV3凋亡时引起了细胞内Ca2+升高,从而介导内质网凋亡信号通路。该研究结果将进一步揭示ns脉冲诱导凋亡的机制。     

ns脉冲电场诱导裸小鼠皮下人黑色素移植瘤凋亡

为研究ns脉冲电场对在体肿瘤的诱导凋亡效应,以接种人黑色素瘤细胞A375的BALB/c裸小鼠为对象,采用电场强度20kV/cm、脉冲宽度300ns的脉冲电场处理后,抑瘤效应宏观观察到肿瘤组织的生长受到显著抑制(P<0.01),透射电子显微镜观察到典型的凋亡细胞超微结构形态,免疫荧光染色法定性检测到caspase-3蛋白表达明显增强,用RT-PCR方法定量检测到caspase-3 mRNA表达明显增强(P<0.01)。实验结果证实了ns脉冲电场能有效抑制在体肿瘤组织生长,机制在于其通过活化肿瘤细胞caspase-3蛋白酶而诱导肿瘤细胞调亡,为ns脉冲电场治疗肿瘤提供了依据。     

高频纳秒脉冲串作用下皮肤肿瘤热效应的多参数有限元仿真与实验

为综合传统微秒脉冲电场和纳秒脉冲电场治疗肿瘤的优点,提出一种高频纳秒脉冲串的脉冲形式来治疗肿瘤。研究此高频纳秒脉冲串在生物组织中产生的热效应,并通过多参数有限元方法对施加镊子电极的皮肤黑色素肿瘤的温度以及热损伤进行仿真。施加的电场强度范围为1~10k V/cm,脉宽范围为50~500ns,脉冲串内重复频率介于100k Hz和1MHz之间,一串脉冲的长度为100μs,脉冲串重复频率为1Hz。对于皮肤肿瘤,施加电场强度5k V/cm、脉宽500ns、脉冲串内重复频率1MHz的脉冲,1s后肿瘤的最高温度仅为37.4℃,热损伤可以忽略不计。对不同电场强度、脉宽、重复频率下皮肤黑色素肿瘤的温度和热损伤进行仿真计算,研究肿瘤温度以及热损伤与脉冲参数的关系,并确定使肿瘤不发生热损伤的脉冲参数范围。对裸鼠在体黑色素肿瘤的温度进行实测,肿瘤的温升结果与仿真中的平均温度变化较为一致。     

Effects of nanosecond pulsed electric fields on the human prostate cancer cell line LNCaP

The effects of nanosecond pulsed electric fields (nsPEF) on the human prostate cancer cell line LNCaP was investigated for changes in protein and DNA concentrations as well as for the 11βHydroxysteroid Dehydrogenase type 2 (11βHSD2) enzyme activity. Electric fields with intensities of 32.5 kV/ cm and pulse widths of 60 ns were applied to cancer cells in trains of 1, 5, and 10 pulses. Protein concentrations were determined through a Bio-Rad DC protein assay, while Hoechst 33258 was used to quantify DNA concentrations. The analyses showed that protein concentrations decreased by 18.77% and 19.04% for 1 and 10 pulses, respectively, while DNA concentrations decreased by 5.29%, 8.95%, and 18.36% for 1, 5, and 10 pulses, respectively, as compared to control groups. The 11βHSD2 enzyme activity in LNCaP cells also decreased following exposure, thus supporting our initial hypothesis that ultrashort pulses affect intracellular structures; thus, a decrease in the enzyme activity could result in enhancing the anti-proliferative actions of glucocorticoids.     

ns脉冲电场发生器的研制及应用

为了研究ns脉冲电场诱导肿瘤细胞的凋亡效应,结合开关电源技术和脉冲功率技术,研制了一套多参数大范围独立可调的ns脉冲电场发生器。该发生器由高压直流电源、脉冲形成电路和辅助电路3部分组成,输出的脉冲幅值0～1.2kV内连续可调,重复频率1Hz～10kHz内连续可调,脉冲宽度分100、200、600和1000ns4级可调,脉冲个数可以任意预置,上升时间<30ns,并具有显示和保护功能。实验室调试和医学细胞实验结果表明,该发生器调节方便、性能稳定,能够有效诱导人肝癌细胞凋亡,为ns脉冲电场诱导肿瘤细胞凋亡的机理和阈值参数选择规律的深入研究奠定了基础。     

Changes in electrical impedance of biological matter due to the application of ultrashort high voltage pulses

Time domain-based impedance measurements were used to study the changes in electrical parameters of biological samples following the application of ultrashort high voltage (HV) pulses. Pulses with very short duration (300 ns) caused a significant drop in post pulse resistance of the plasma membrane only at high field strengths in excess of 20 kV/cm. The conductivity of the plasma membrane returned to almost pre-pulse values within less than 10 ms after the field was applied. Further steps of recovery, attributed to pore shrinking and resealing with an exponential decay of the conductivity, as is expected in electroporation, were not observed. An increase in medium conductance, as recorded minutes after the pulse, arises mainly from cell damage. Although pore formation is a possible effect of the high electric field, our results suggest further disturbance of the membrane-like micelle formation or even the creation of large defects, forced by mechanical tension within the cell membrane.     

Biological effects of narrow band pulsed electric fields

This paper describes the process of narrow band pulsed electric fields (NPEFs) and its effect on mammalian cells. The NPEF consists of a pulse modulated sinusoidal wave (PMSW), which allows delivery of well-defined electric fields in terms of frequency, field strength and deposition energy to the biological systems. 100 mus long sinusoidal electric fields with a frequency of 0.02, 2 or 50 MHz and field strengths of up to 2 kV/cm are applied to CHO cells with variation in the DNA density in the cells investigated by means of Acridine Orange assay. The experiments indicate that 50 MHz fields cause DNA degradation without cell membrane defects, while 0.02 MHz fields lead to an increase in membrane permeability which is similar to the effect known as electroporation. The intermediate frequency of 2 MHz influences both the membrane and DNA. It is demonstrated that the MHz range narrowband electric fields with the amplitude level of 1 kV/cm cause intracellular effects in mammalian cells.     

Nanosecond High Voltage Pulse Generator Using Water Gap Switch for Compact High Power Pulsed Microwave Generator

Nanosecond and sub-nanosecond high voltage pulses can provide new applications. A cancer treatment by an ultra-short pulse high electric field is one of them. High power pulsed microwave has been proposed to apply the high electric field for that treatment. This work focuses on the design of a compact high power pulsed microwave generator using a nanosecond pulse power generator for the cancer treatment. To obtain fast rise time of voltage and current for nanosecond pulses, a switch has to have low inductance. Water gap switches have this property. Since water has a dielectric strength exceeding 1 MV/cm, gap distances for switching can be reduced to several hundreds of micrometers, and still allow switching of tens of kV. The narrow gaps allow us to reduce the switch inductance. In this study, the water gap switch was built in a Blumlein pulse forming line. The Blumlein line was designed to provide a pulse of 1 ns and to match an impedance to 16 Omega of an antenna. By using the water gap switch in the Blumlein line, the voltage rise time obtained approximately 750 ps at 13 kV of a peak pulse voltage. An electromagnetic wave was radiated underwater by the loop antenna. A measuring antenna at 0.15 m and 0.4 m from the radiating antenna caught electric field intensities of 116 kV/m and 32 kV/m, respectively. A frequency element of 250 MHz had higher intensity.     

Aspects of lipid membrane bio-responses to subnanosecond, ultrahigh voltage pulsing

Membrane electroporation is probably one of the best-known effects of applying external voltages to biological cells. Reports in the literature have focused on relatively long voltage pulse durations (100 ns-1 ms). Here we probe the very short (< 1 ns), but intense electric field (> 500 kV/cm) regime that is made possible by advances in pulsed power technology. Our analyses based on continuum Smoluchowski and Molecular Dynamics (MD) approaches, predict two new aspects. First, it is shown that pore formation rates would be dramatically lower than predicted by conventional theory due to their dependence on local pore area. Second, such high fields are predicted to affect membrane proteins and ion-channels, without causing electroporation in regions between the proteins. Hence, such high voltage, short duration pulsing should not be associated with electroporation alone, but rather be viewed as a novel vehicle that opens possibilities for a range of new electrically-driven bio-response phenomena.     

基于现场可编程门阵列的全固态高压ns脉冲发生器

为研究ns脉冲电场诱导肿瘤细胞的凋亡效应,结合Marx发生器原理和全固态开关技术,研制了一套基于现场可编程门阵列(FPGA)的多参数可调全固态高压ns脉冲发生器。该发生器主要包括高压直流电源、Marx电路、FPGA控制电路和负载4部分。Marx电路采用金属氧化物半导体场效应晶体管(MOSFET)作为控制开关代替传统的火花间隙开关,用二极管代替电阻。FPGA产生多路同步触发脉冲信号,通过光纤进行隔离后可作为MOSFET的原始控制信号,同步驱动多个MOSFET。FPGA控制电路控制充电电压和输出脉冲的宽度、频率,并具有保护功能。实验结果表明,该脉冲发生器可产生幅值(0～8kV)连续可调、脉宽(200～1 000ns)灵活可变、频率(1～1 000Hz)独立可控,前沿35ns的高压ns脉冲,为进一步探索ns脉冲电场生物医学效应奠定了基础。     

Load Section Design of a Pulsed Power Generator for X-pinch

The load section of a pulsed power generator for X-pinch was designed. It is a work that repeats itself in cycles as follows: making small changes in size and configuration of the electrode system in order to reduce inductance rarr calculating the inductance rarr calculating the load voltage with the measured forward going voltage wave along the pulse transmission line (PTL) incident upon the load rarr calculating the electric field rarr checking if any surface flashover or breakdown in vacuum happens. In the final designed load section, the highest field on the surface of the insulator, a plexiglass diaphragm, is 113 kV/cm and the highest field in vacuum is 298 kV/cm, respectively lower than the surface flashover voltage and the vacuum breakdown voltage in case of an applied pulse voltage of 100 ns of full width at half maximum (FWHIVI) and 500 kV in amplitude.     

Bacterial decontamination of liquids with pulsed electric fields

The effect of pulsed electric fields on the viability of microorganisms, mainly bacteria, in liquids has been studied since the 1960's. Experimental results obtained over a large range of electrical and microbiological parameters, point towards an irreversible formation of pores in the cell membrane as the mechanism for lysing. The model of membrane pore formation seems to fail only for ultrashort electrical pulses, where intracellular effects, and possibly resonant effects, might dominate. This paper presents an overview of the effect of pulsed electric fields on the viability of microorganisms in liquids, In particular, the lytic effect of variations in the electrical pulse parameters, such as pulse shape, amplitude, duration, and single shot vs. repetitive operation, is described, A major application of the pulsed electric field method is `cold' bacterial decontamination of liquid food and drinking water. The energy consumption for complete bacterial decontamination is presently 100 to 400 kJ/l. A possible reduction of the required energy by utilizing intracellular electric field interactions and resonance effects is discussed     

Inactivation of microorganisms by pulsed electric fields ofdifferent voltage waveforms

Processing foods with HV pulsed electric fields (PEF) is a new technology to inactivate microorganisms and denature enzymes with only a small increase in temperature. Introduction of this new technology will replace or complement conventional thermal processing methods. It will also provide consumers with safe, nutritious foods with fresh quality. For a given peak value of field intensity and amount of electric energy input, PEF inactivation of microorganisms is closely related to the waveform of applied pulses. Inactivation of microorganisms was studied with different waveshapes including exponential decay, oscillatory decay, square waves, and bipolar pulses. Microbial inactivation was tested in a parallel-plate static treatment chamber. Treatment field intensity ranged from 12 to 40 kV/cm while pulse length ranged from 30 to 180 μs. From the microbial test results, bipolar square-wave pulses are the most efficient in terms of microbial inactivation for commercial PEF pasteurization     

Heat conduction in microbes exposed to pulsed electric fields

Nonthermal pasteurization of liquid foods by intense pulsed electric fields results in advantages over conventional heat pasteurization. Thermal damage to liquid foods can adversely affect the flavor and taste and result in loss of nutrients. Numerical modeling of heating parameters near the microbe during exposure to these intense electric fields is described. Boundary conditions at membrane interfaces included the continuity of temperature and continuity of heat flow. Temperature, heat flow vector, and heat source were included in the one-dimensional model. Two simulations are reported here. Simulation 1 consisted of a 0.5 μm wide microbe suspended in liquid with conductivity of 0.01 S/m and was treated with an applied electric field of 40 kV/cm for 375 ns. Simulation 2 contained a 1.0 μm wide microbe suspended in liquid with conductivity of 0.1 S/m and was treated with an applied electric field of 40 kV/cm for 1 μs. Comparison is made with a uniform conductivity model (ohmic), and it is shown that significant differences exist in the heating parameters between the two models. For the parameters used in these one-dimensional simulations, conditions for electroporation were found to exist without a significant temperature rise in the microbe     

Diode Opening Switch Based Nanosecond High Voltage Pulse Generators for Biological and Medical Applications

Studies of short (few ns) pulse cell electroperturbation require high-voltage nanosecond pulses delivered to low-impedance (10 Omega) electroporation cuvette loads. Here we present two pulse generators for these applications. The generators comprise of three stages of pulse modulation: an LC resonant stage switched by IGBT/MOSFET; a magnetic pulse compression stage; and a diode opening switch output stage. The all-solid-state design results in reproducible pulses and reliable, long-life operation. Pulses produced by the LC resonant stage are compressed using nonlinear properties of the magnetic material. The compressed pulses are fed to the diode opening switch, which is constructed by series and parallel connecting ordinary automotive rectifying diodes. Two versions of the pulse generator are described: The first version is capable of generating 20 ns wide, 4.5 kV amplitude pulses of 20 Hz repetition rate. A second version generates 5 ns, 7.5 kV amplitude pulses into the same 10 Omega cuvette load.