高强度皮秒脉冲电场致Hela细胞凋亡及机制的实验研究
|
摘要
肿瘤的无创与靶向治疗是世界各国研究者们所面临的医学难题同时也是最具广阔应用前景的研究方向。近年来脉冲电场(Pulsed ElectricField, PEF)的生物医学效应颇受人们的关注。国内外同行的前期研究工作都发现脉冲电场对肿瘤细胞有杀伤效应，并将其初步应用于临床，证实可有效消融肿瘤组织。按脉宽的不同，脉冲电场可分为毫秒（ms）、微秒（s）、纳秒（ns）、皮秒（ps）等。研究者们对毫秒、微秒、纳秒脉冲电场进行了较深入的研究，然而毫秒、微秒、纳秒脉冲的导入需要借助有创或微创的电极穿刺至肿瘤组织，这就在一定程度上限制了该技术的临床应用。因此，寻求一种能无创地进入体内深部组织的脉冲电场以实现肿瘤的无创治疗成为新的研究方向。
根据时域电场理论，皮秒脉冲电场具有几乎从直流到高达GHz的丰富的超宽带频谱，因此皮秒脉冲电场具有很高的空间分辨率和时间分辨率，信号失真小。通过冲激脉冲辐射天线（Impulse radiating antenna,IRA）能将皮秒脉冲电场无创地传导至深部组织并形成靶点的聚焦，同时能避免对正常组织造成的损伤，从而可以实现肿瘤的无创治疗。但目前皮秒脉冲电场对肿瘤的作用及机理尚处于理论研究阶段，关于其对肿瘤的生物学效应的实验研究鲜有报道。
脉冲电场对细胞的作用具有一定的窗口效应，也就是脉冲电场的参数选择不同，细胞的作用靶点会发生相应的变化。当施加脉冲电场的脉宽从毫秒降低到微秒，再到纳秒，所作用的细胞靶点也会由细胞膜转移到细胞质及核质，所产生的作用也由诱导基因转染，诱导电穿孔、不可逆电击穿，到诱导细胞的内处理。如果脉冲电场的脉宽进一步降低到亚纳秒甚至皮秒范围，理论上讲对细胞的作用将转移到线粒体等细胞器膜。那么在皮秒级脉宽采用高强度的脉冲电场，将导致线粒体的跨膜电位发生变化，从而可以通过线粒体途径诱导细胞发生凋亡。
宫颈癌是最常见的妇科恶性肿瘤之一。针对目前宫颈癌发病的早期化、年轻化，对治疗提出了新的要求。传统的手术治疗，往往会导致对生殖器官（阴道、宫颈和子宫）的严重损害，影响性功能和生育能力。鉴于皮秒脉冲电场靶向聚焦的治疗处于早期基础研究阶段，我们希望选择体表的肿瘤进行相关研究。宫颈虽然不在体表，但是可以通过简单的器械充分暴露。同时，宫颈癌被称为―善良的‖癌症，从早期的癌前病变发展到真正的恶性病变存在一个较长的―窗口期‖，在不同的患者中这个时间段分别约为数年至十余年之久。通过常规的筛查手段，宫颈癌完全可以在早期就被发现确诊，这就为脉冲电场治疗提供了非常适合的时机。
本课题以人宫颈癌Hela细胞为研究对象，以高强度皮秒脉冲电场为研究手段，采用Caspase活性测定试剂盒、激光共聚焦扫描显微镜、免疫细胞化学、Western blot，RT-PCR (Real time polymerase chainreaction)等方法检测细胞凋亡以及线粒体凋亡相关蛋白和基因表达。从分子、细胞水平深入研究高强度皮秒脉冲电场的细胞靶点是否可作用于线粒体，并通过线粒体途径致肿瘤细胞凋亡，以期为皮秒脉冲的治疗提供初步依据，推动其尽早进入临床，为宫颈癌患者提供一种无创治疗的保留生育功能的新方法。
目的：探讨高强度皮秒脉冲电场作用于Hela细胞，凋亡相关因子Caspase-3及Caspase-9活性及基因表达的改变。
方法：将HeLa细胞分为对照组和不同剂量高强度皮秒脉冲电场处理组。脉冲电场处理后6h、12h,按Caspase活性测定试剂盒进行操作，酶标仪测定Caspase-3，Caspase-9蛋白活性；脉冲电场处理后2h、6h，RT-PCR法检测Caspase-3，Caspase-9基因转录强度，分析脉冲电场处理后Caspase-3，Caspase-9活性及基因表达的变化。
结果：酶标仪检测结果显示，脉冲电场处理后Caspase-3，Caspase-9蛋白活性升高，呈现剂量依赖性，随脉冲个数增加，活性增强，随场强增加，活性增强；各处理组较对照组相比差异有统计学意义（p<0.01）。RT-PCR结果发现，脉冲电场处理后Caspase-3，Caspase-9基因转录水平上升，呈剂量效应关系，随脉冲个数增加，转录水平升高；各处理组与对照组相比，差异有统计学意义（p<0.05）。Caspase-3，Caspase-9蛋白活性6h、12h时间组间差异无统计学意义（p>0.05）。Caspase-3，Caspase-9基因转录强度6h组较2h组升高，差异有统计学意义（p<0.05）。可见Caspase-3，Caspase-9活性于脉冲电场作用2h显著上升，6h可达最大效应。
结论：高强度皮秒脉冲电场作用于HeLa细胞，线粒体凋亡相关分子Caspase-3，Caspase-9被活化，呈剂量效应关系，随着脉冲个数的增加，Caspase的活性增强，随着场强的增加，Caspase的活性增强。初步证实了高强度皮秒脉冲电场可活化Caspase-3，Caspase-9，诱导细胞凋亡。
目的：探讨高强度皮秒脉冲电场作用于Hela细胞，线粒体途径凋亡的发生及其机制。
方法：将HeLa细胞分为对照组和4个不同剂量高强度皮秒脉冲电场处理组。脉冲电场处理后即刻用Fluo-3/AM标记细胞内Ca~(2+)的浓度[Ca~(2+)]i，应用激光共聚焦显微镜观察Hela细胞内Fluo-3/AM荧光探针的平均荧光强度，并做半定量分析。脉冲电场处理后2h、6h、12h，用荧光探针罗丹明123（Rhodamine123, Rh123）标记线粒体跨膜电位，应用激光共聚焦显微镜观察Hela细胞内Rh123荧光探针的平均荧光强度，并做半定量分析，探讨高强度皮秒脉冲处理后细胞内Ca~(2+)浓度和线粒体跨膜电位的变化。脉冲电场处理后2h、6h，Western blot法检测线粒体释放到细胞浆分子细胞色素C (cytochrome C，Cyt c)、凋亡诱导因子(apoptotic inducing factor，AIF)蛋白水平的表达。
结果：高强度皮秒脉冲电场作用后，细胞内Ca~(2+)浓度[Ca~(2+)]i升高，呈现剂量依赖性，随脉冲个数增加，Ca~(2+)的浓度升高，各处理组与对照组相比，差异有统计学意义（p<0.01）。脉冲电场处理后线粒体跨膜电位降低，呈剂量效应关系，随脉冲个数增加，跨膜电位降低，各处理组与对照组相比，差异有统计学意义（p<0.01）。2h，6h时间组差异有统计学意义（p<0.05）。6h，12h时间组差异无统计学意义（p>0.05），说明线粒体跨膜电位在处理后2小时下降，6小时进一步下降，达最大效应。Western blot结果显示处理组细胞色素C及凋亡诱导因子蛋白水平升高，呈现剂量依赖性，随着脉冲个数增加，蛋白水平升高，各处理组与对照组相比差异有统计学意义（p<0.05）。2h、6h时间组差异有统计学意义（p<0.05）。
结论：高强度皮秒脉冲电场可作用于细胞线粒体，导致线粒体跨膜电位变化，释放促凋亡因子Cyt c，AIF及钙离子，通过线粒体途径介导细胞凋亡。
目的：探讨高强度皮秒脉冲电场作用于Hela细胞，细胞凋亡的调控分子表达的改变。
方法：将HeLa细胞分为对照组和不同剂量高强度皮秒脉冲电场处理组。脉冲电场处理后免疫细胞化学观察Bcl-2家族Bax, Bcl-2蛋白水平的表达。脉冲电场处理后2h、6h，RT-PCR检测线粒体凋亡途径调控分子p53及Bcl-2家族Bax, Bcl-2基因水平的表达。
结果：脉冲电场处理后，免疫细胞化学检测Bax：对照组细胞胞浆呈棕黄色，胞核染色浅淡；处理组细胞的染色形态与分布和对照组相似，但着色更深。对照组和处理组平均灰度值分别为86.27±9.32和110.75±10.23,两组差异有统计学意义(P<0.05)。免疫细胞化学检测Bcl-2：对照组细胞呈阳性表达，即胞浆呈棕黄色，部分细胞核也染色；处理组染色形态同对照组细胞，但染色强度减弱。对照组和处理组平均灰度值分别为102.36±10.45和69.45±9.38，两组差异有统计学意义(P<0.05)。Bax, p53基因转录强度处理组较对照组升高，Bcl-2较对照组降低，差异有统计学意义（p<0.05），呈剂量效应关系，Bax, p53基因转录强度与脉冲个数呈正相关，Bcl-2与脉冲个数呈负相关。6h组与2h组相比，差异有统计学意义（p<0.05）。
结论：高强度皮秒脉冲作用于Hela细胞，凋亡调控相关分子Bax表达升高，Bcl-2表达降低，Bax与Bcl-2比例失衡，p53表达升高，表明Bcl-2家族与p53在脉冲电场诱导的Hela细胞凋亡发生中起到了一定的作用。
Targeted non-invasive treatment of tumor is the world's mostpromising area of medical research. The application of pulsed electricfields (PEF) is emerging as a new technique for tumor therapy. Accordingto the pulse duration, PEF can be classified into millisecond (ms),microsecond (s), nanosecond (ns), picosecond, et al. Most researchersfocused on the ms, s and ns pulse range for a more in-depth study.However, the application of ms, s and ns PEF still needs to use theinvasive or minimally invasive needle or plate electrodes, to guide thepuncture of tumor tissue, which to some extent limit the clinical applicationof this method.
Picosecond PEF (psPEF) has a wealth of ultra-broadband spectrum,with a high time and spatial resolution, and low signal distortion. It can betransferred to target deep tissue non-invasively and precisely withwideband antennas. But to our knowledge, the research of the biologicaleffect of psPEF on cells remains rare.
Electric field possesses parameters related different biophysical effects, that is, the impact of electric field pulses on cells has a certain windoweffect. Ms or s PEF targets outer membrane mainly, and there is littleinfluence to cell nucleus, mitochondrion, and other organelles; thus, itcauses electroporation to the outer membrane. As the pulse durationdecreases, the electroporation effect changes gradually from the outermembrane to intracellular organelle membrane. Submicrosecond PEF caninduce significant voltages across both the inner and outer membranes,therefore, causing damage to both the inner and outer membranes.
While these effects of PEF continue to be explored, a new domain ofpulsed electric field interactions with cell structures and functions opens upwhen the pulse duration is reduced to values such that membrane chargingbecomes negligible. For mammalian cells, this holds for a pulse duration ofone nanosecond or less. We dare to assume from the rules above, when theelectrical pulses duration is shorter than one nanosecond, PEF can inducelarger voltage across the inner membrane and acts mostly on intracellularsubstructures. According to cell biology and electromagnetic theory, themitochondrial membrane charging time constant (a few hundreds ofpicoseconds) is much shorter than nuclear membrane and the membranecharging time constant (tens of nanoseconds, and hundreds of nanoseconds,respectively). Under the action of the intense psPEF, the mitochondrialmembrane will charge fast, at a time when the nuclear membrane and themembrane had no chance to respond, so the mitochondria transmembrane potential will be changed. We can speculate that intense psPEF target themitochondria and lead to changes in transmembrane potential, release ofCyt c and AIF etc., activation of caspase9and caspase3, and finallyapoptosis.
Cervical cancer is one of the most common gynecologicalmalignancies. Its incidence in young women is increasing in recent years.The traditional surgical treatment often leads to genital tract severe damageand affect patients’sexual function and fertility. Despite of advances insurgical techniques, conservative treatment such as radical trachelectomyhas appeared, it still has a great impact on patients’fertility. Non-invasivetreatment with preserved fertility is the expectation for both doctors andpatients.
In this study, we tested the hypothesis that intense psPEF can inducecell apoptosis through mitochondrial path. Human cervical cancer cells,Hela cells were chosen to be exposed to psPEF. This research can not onlyenrich the biological effects of picosecond pulsed field theory, but alsoprovide cervical cancer patients a new non-invasive treatment of preservingfertility.
Objective: To investigate the apoptosis-related factors Caspase-3and caspase-9activity and gene expression in HeLa cells after exposed tointense picosecond pulsed electric fields.
Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups.6hours and12hours after exposed to intense psPEF, Caspase protein activity kit wasused to test the Caspase-3and Caspase-9activity.2hours and6hours afterexposed to intense psPEF, RT-PCR was used to test the Caspase-3andCaspase-9gene expression.
Results: Intense picosecond PEF could significantly enhanced theCaspase-3and Caspase-9activity and gene expression in a dose-dependentmanner, the protein activity increased in parallel with pulse numbers andelectric field amplitude. Each treated group showed significant difference incomparison to the control group (all P<0.05). The caspase activity increased2hours after pulses and could reach a maximum level6hours afterexposed to intense psPEF.
Conclusion: psPEF could generate activation of caspase-3andcaspase-9in Hela cells, and the effect was in a dose-dependent manner. Theactivity increased in parallel with pulse numbers and electric fieldamplitude.
Objective: To investigate the Mechanism of apoptosis in Hela cellsthrough a mitochondrial mediated pathway induced by intense psPEF.
Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups. Aftertreated with psPEF of different doses,[Ca2+]i was marked by Fluo3-AM andanalyzed by laser confocal scanning microscope.2h,6h, and12h afterexposed to intense psPEF, mitochondrial membrane potential was markedwith Rh123and detected by laser confocal scanning microscope.2h,6h afterexposed to intense psPEF, Western blot was used to measure the proteinlevels of Cyt C and AIF.
Results:[Ca2+]i elevated significantly shortly after treated with psPEF.Mitochondrial membrane potential declined2hours after pulses, andreached a minimum level6hours after pulses. The protein expression ofCyt C and AIF increased significantly2hours after pulses. The effect wasin a dose-dependent manner. Each treated group showed significantdifference in comparison to the control group (all P<0.05).
Conclusion: Intense psPEF could target the mitochondria and lead tochanges in transmembrane potential, release of Cyt C and AIF and inducecells apoptosis through mitochondrial pathway.
Objective: To study the effects of intense psPEF on regulation ofapoptosis in Hela cells.
Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups.Immunocytochemistry was used to observe the bax and bcl-2proteinexpression after exposed to psPEF.2h,6h after exposed to intense psPEF,the mRNA expression of bax, bcl-2and p53was detected with RT-PCR.
Results: Immunocytochemistry of Bax: the cytoplasm of the controlgroup was stained brown, and the treated cells showed the same distribution,but were stained more deeply. The average level of the control group andtreatment group were86.27±9.32and110.75±10.23individually, thedifference was statistically significant (P<0.05). Immunocytochemistry ofBcl-2: the cytoplasm of the control group was stained brown, and treatedcells showed the same distribution, but were stained less deeply. Theaverage level of the control group and treatment group were102.36±10.45and69.45±9.38individually, the difference was statistically significant(P<0.05).The mRNA expression of bax and p53increased significantly2hours after pulses, and bcl-2decreased2hours after pulses. The effect wasin a dose-dependent manner. Each treated group showed significantdifference in comparison to the control group (all P<0.05).
Conclusion: Imbalance of Bax and Bcl-2, p53were involved in theprocess of apoptosis induced by intense psPEF.
根据时域电场理论，皮秒脉冲电场具有几乎从直流到高达GHz的丰富的超宽带频谱，因此皮秒脉冲电场具有很高的空间分辨率和时间分辨率，信号失真小。通过冲激脉冲辐射天线（Impulse radiating antenna,IRA）能将皮秒脉冲电场无创地传导至深部组织并形成靶点的聚焦，同时能避免对正常组织造成的损伤，从而可以实现肿瘤的无创治疗。但目前皮秒脉冲电场对肿瘤的作用及机理尚处于理论研究阶段，关于其对肿瘤的生物学效应的实验研究鲜有报道。
脉冲电场对细胞的作用具有一定的窗口效应，也就是脉冲电场的参数选择不同，细胞的作用靶点会发生相应的变化。当施加脉冲电场的脉宽从毫秒降低到微秒，再到纳秒，所作用的细胞靶点也会由细胞膜转移到细胞质及核质，所产生的作用也由诱导基因转染，诱导电穿孔、不可逆电击穿，到诱导细胞的内处理。如果脉冲电场的脉宽进一步降低到亚纳秒甚至皮秒范围，理论上讲对细胞的作用将转移到线粒体等细胞器膜。那么在皮秒级脉宽采用高强度的脉冲电场，将导致线粒体的跨膜电位发生变化，从而可以通过线粒体途径诱导细胞发生凋亡。
宫颈癌是最常见的妇科恶性肿瘤之一。针对目前宫颈癌发病的早期化、年轻化，对治疗提出了新的要求。传统的手术治疗，往往会导致对生殖器官（阴道、宫颈和子宫）的严重损害，影响性功能和生育能力。鉴于皮秒脉冲电场靶向聚焦的治疗处于早期基础研究阶段，我们希望选择体表的肿瘤进行相关研究。宫颈虽然不在体表，但是可以通过简单的器械充分暴露。同时，宫颈癌被称为―善良的‖癌症，从早期的癌前病变发展到真正的恶性病变存在一个较长的―窗口期‖，在不同的患者中这个时间段分别约为数年至十余年之久。通过常规的筛查手段，宫颈癌完全可以在早期就被发现确诊，这就为脉冲电场治疗提供了非常适合的时机。
本课题以人宫颈癌Hela细胞为研究对象，以高强度皮秒脉冲电场为研究手段，采用Caspase活性测定试剂盒、激光共聚焦扫描显微镜、免疫细胞化学、Western blot，RT-PCR (Real time polymerase chainreaction)等方法检测细胞凋亡以及线粒体凋亡相关蛋白和基因表达。从分子、细胞水平深入研究高强度皮秒脉冲电场的细胞靶点是否可作用于线粒体，并通过线粒体途径致肿瘤细胞凋亡，以期为皮秒脉冲的治疗提供初步依据，推动其尽早进入临床，为宫颈癌患者提供一种无创治疗的保留生育功能的新方法。
目的：探讨高强度皮秒脉冲电场作用于Hela细胞，凋亡相关因子Caspase-3及Caspase-9活性及基因表达的改变。
方法：将HeLa细胞分为对照组和不同剂量高强度皮秒脉冲电场处理组。脉冲电场处理后6h、12h,按Caspase活性测定试剂盒进行操作，酶标仪测定Caspase-3，Caspase-9蛋白活性；脉冲电场处理后2h、6h，RT-PCR法检测Caspase-3，Caspase-9基因转录强度，分析脉冲电场处理后Caspase-3，Caspase-9活性及基因表达的变化。
结果：酶标仪检测结果显示，脉冲电场处理后Caspase-3，Caspase-9蛋白活性升高，呈现剂量依赖性，随脉冲个数增加，活性增强，随场强增加，活性增强；各处理组较对照组相比差异有统计学意义（p<0.01）。RT-PCR结果发现，脉冲电场处理后Caspase-3，Caspase-9基因转录水平上升，呈剂量效应关系，随脉冲个数增加，转录水平升高；各处理组与对照组相比，差异有统计学意义（p<0.05）。Caspase-3，Caspase-9蛋白活性6h、12h时间组间差异无统计学意义（p>0.05）。Caspase-3，Caspase-9基因转录强度6h组较2h组升高，差异有统计学意义（p<0.05）。可见Caspase-3，Caspase-9活性于脉冲电场作用2h显著上升，6h可达最大效应。
结论：高强度皮秒脉冲电场作用于HeLa细胞，线粒体凋亡相关分子Caspase-3，Caspase-9被活化，呈剂量效应关系，随着脉冲个数的增加，Caspase的活性增强，随着场强的增加，Caspase的活性增强。初步证实了高强度皮秒脉冲电场可活化Caspase-3，Caspase-9，诱导细胞凋亡。
目的：探讨高强度皮秒脉冲电场作用于Hela细胞，线粒体途径凋亡的发生及其机制。
方法：将HeLa细胞分为对照组和4个不同剂量高强度皮秒脉冲电场处理组。脉冲电场处理后即刻用Fluo-3/AM标记细胞内Ca~(2+)的浓度[Ca~(2+)]i，应用激光共聚焦显微镜观察Hela细胞内Fluo-3/AM荧光探针的平均荧光强度，并做半定量分析。脉冲电场处理后2h、6h、12h，用荧光探针罗丹明123（Rhodamine123, Rh123）标记线粒体跨膜电位，应用激光共聚焦显微镜观察Hela细胞内Rh123荧光探针的平均荧光强度，并做半定量分析，探讨高强度皮秒脉冲处理后细胞内Ca~(2+)浓度和线粒体跨膜电位的变化。脉冲电场处理后2h、6h，Western blot法检测线粒体释放到细胞浆分子细胞色素C (cytochrome C，Cyt c)、凋亡诱导因子(apoptotic inducing factor，AIF)蛋白水平的表达。
结果：高强度皮秒脉冲电场作用后，细胞内Ca~(2+)浓度[Ca~(2+)]i升高，呈现剂量依赖性，随脉冲个数增加，Ca~(2+)的浓度升高，各处理组与对照组相比，差异有统计学意义（p<0.01）。脉冲电场处理后线粒体跨膜电位降低，呈剂量效应关系，随脉冲个数增加，跨膜电位降低，各处理组与对照组相比，差异有统计学意义（p<0.01）。2h，6h时间组差异有统计学意义（p<0.05）。6h，12h时间组差异无统计学意义（p>0.05），说明线粒体跨膜电位在处理后2小时下降，6小时进一步下降，达最大效应。Western blot结果显示处理组细胞色素C及凋亡诱导因子蛋白水平升高，呈现剂量依赖性，随着脉冲个数增加，蛋白水平升高，各处理组与对照组相比差异有统计学意义（p<0.05）。2h、6h时间组差异有统计学意义（p<0.05）。
结论：高强度皮秒脉冲电场可作用于细胞线粒体，导致线粒体跨膜电位变化，释放促凋亡因子Cyt c，AIF及钙离子，通过线粒体途径介导细胞凋亡。
目的：探讨高强度皮秒脉冲电场作用于Hela细胞，细胞凋亡的调控分子表达的改变。
方法：将HeLa细胞分为对照组和不同剂量高强度皮秒脉冲电场处理组。脉冲电场处理后免疫细胞化学观察Bcl-2家族Bax, Bcl-2蛋白水平的表达。脉冲电场处理后2h、6h，RT-PCR检测线粒体凋亡途径调控分子p53及Bcl-2家族Bax, Bcl-2基因水平的表达。
结果：脉冲电场处理后，免疫细胞化学检测Bax：对照组细胞胞浆呈棕黄色，胞核染色浅淡；处理组细胞的染色形态与分布和对照组相似，但着色更深。对照组和处理组平均灰度值分别为86.27±9.32和110.75±10.23,两组差异有统计学意义(P<0.05)。免疫细胞化学检测Bcl-2：对照组细胞呈阳性表达，即胞浆呈棕黄色，部分细胞核也染色；处理组染色形态同对照组细胞，但染色强度减弱。对照组和处理组平均灰度值分别为102.36±10.45和69.45±9.38，两组差异有统计学意义(P<0.05)。Bax, p53基因转录强度处理组较对照组升高，Bcl-2较对照组降低，差异有统计学意义（p<0.05），呈剂量效应关系，Bax, p53基因转录强度与脉冲个数呈正相关，Bcl-2与脉冲个数呈负相关。6h组与2h组相比，差异有统计学意义（p<0.05）。
结论：高强度皮秒脉冲作用于Hela细胞，凋亡调控相关分子Bax表达升高，Bcl-2表达降低，Bax与Bcl-2比例失衡，p53表达升高，表明Bcl-2家族与p53在脉冲电场诱导的Hela细胞凋亡发生中起到了一定的作用。
Targeted non-invasive treatment of tumor is the world's mostpromising area of medical research. The application of pulsed electricfields (PEF) is emerging as a new technique for tumor therapy. Accordingto the pulse duration, PEF can be classified into millisecond (ms),microsecond (s), nanosecond (ns), picosecond, et al. Most researchersfocused on the ms, s and ns pulse range for a more in-depth study.However, the application of ms, s and ns PEF still needs to use theinvasive or minimally invasive needle or plate electrodes, to guide thepuncture of tumor tissue, which to some extent limit the clinical applicationof this method.
Picosecond PEF (psPEF) has a wealth of ultra-broadband spectrum,with a high time and spatial resolution, and low signal distortion. It can betransferred to target deep tissue non-invasively and precisely withwideband antennas. But to our knowledge, the research of the biologicaleffect of psPEF on cells remains rare.
Electric field possesses parameters related different biophysical effects, that is, the impact of electric field pulses on cells has a certain windoweffect. Ms or s PEF targets outer membrane mainly, and there is littleinfluence to cell nucleus, mitochondrion, and other organelles; thus, itcauses electroporation to the outer membrane. As the pulse durationdecreases, the electroporation effect changes gradually from the outermembrane to intracellular organelle membrane. Submicrosecond PEF caninduce significant voltages across both the inner and outer membranes,therefore, causing damage to both the inner and outer membranes.
While these effects of PEF continue to be explored, a new domain ofpulsed electric field interactions with cell structures and functions opens upwhen the pulse duration is reduced to values such that membrane chargingbecomes negligible. For mammalian cells, this holds for a pulse duration ofone nanosecond or less. We dare to assume from the rules above, when theelectrical pulses duration is shorter than one nanosecond, PEF can inducelarger voltage across the inner membrane and acts mostly on intracellularsubstructures. According to cell biology and electromagnetic theory, themitochondrial membrane charging time constant (a few hundreds ofpicoseconds) is much shorter than nuclear membrane and the membranecharging time constant (tens of nanoseconds, and hundreds of nanoseconds,respectively). Under the action of the intense psPEF, the mitochondrialmembrane will charge fast, at a time when the nuclear membrane and themembrane had no chance to respond, so the mitochondria transmembrane potential will be changed. We can speculate that intense psPEF target themitochondria and lead to changes in transmembrane potential, release ofCyt c and AIF etc., activation of caspase9and caspase3, and finallyapoptosis.
Cervical cancer is one of the most common gynecologicalmalignancies. Its incidence in young women is increasing in recent years.The traditional surgical treatment often leads to genital tract severe damageand affect patients’sexual function and fertility. Despite of advances insurgical techniques, conservative treatment such as radical trachelectomyhas appeared, it still has a great impact on patients’fertility. Non-invasivetreatment with preserved fertility is the expectation for both doctors andpatients.
In this study, we tested the hypothesis that intense psPEF can inducecell apoptosis through mitochondrial path. Human cervical cancer cells,Hela cells were chosen to be exposed to psPEF. This research can not onlyenrich the biological effects of picosecond pulsed field theory, but alsoprovide cervical cancer patients a new non-invasive treatment of preservingfertility.
Objective: To investigate the apoptosis-related factors Caspase-3and caspase-9activity and gene expression in HeLa cells after exposed tointense picosecond pulsed electric fields.
Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups.6hours and12hours after exposed to intense psPEF, Caspase protein activity kit wasused to test the Caspase-3and Caspase-9activity.2hours and6hours afterexposed to intense psPEF, RT-PCR was used to test the Caspase-3andCaspase-9gene expression.
Results: Intense picosecond PEF could significantly enhanced theCaspase-3and Caspase-9activity and gene expression in a dose-dependentmanner, the protein activity increased in parallel with pulse numbers andelectric field amplitude. Each treated group showed significant difference incomparison to the control group (all P<0.05). The caspase activity increased2hours after pulses and could reach a maximum level6hours afterexposed to intense psPEF.
Conclusion: psPEF could generate activation of caspase-3andcaspase-9in Hela cells, and the effect was in a dose-dependent manner. Theactivity increased in parallel with pulse numbers and electric fieldamplitude.
Objective: To investigate the Mechanism of apoptosis in Hela cellsthrough a mitochondrial mediated pathway induced by intense psPEF.
Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups. Aftertreated with psPEF of different doses,[Ca2+]i was marked by Fluo3-AM andanalyzed by laser confocal scanning microscope.2h,6h, and12h afterexposed to intense psPEF, mitochondrial membrane potential was markedwith Rh123and detected by laser confocal scanning microscope.2h,6h afterexposed to intense psPEF, Western blot was used to measure the proteinlevels of Cyt C and AIF.
Results:[Ca2+]i elevated significantly shortly after treated with psPEF.Mitochondrial membrane potential declined2hours after pulses, andreached a minimum level6hours after pulses. The protein expression ofCyt C and AIF increased significantly2hours after pulses. The effect wasin a dose-dependent manner. Each treated group showed significantdifference in comparison to the control group (all P<0.05).
Conclusion: Intense psPEF could target the mitochondria and lead tochanges in transmembrane potential, release of Cyt C and AIF and inducecells apoptosis through mitochondrial pathway.
Objective: To study the effects of intense psPEF on regulation ofapoptosis in Hela cells.
Methods: HeLa cells were divided into control group and differentdoses of intense picosecond pulsed electric fields treated groups.Immunocytochemistry was used to observe the bax and bcl-2proteinexpression after exposed to psPEF.2h,6h after exposed to intense psPEF,the mRNA expression of bax, bcl-2and p53was detected with RT-PCR.
Results: Immunocytochemistry of Bax: the cytoplasm of the controlgroup was stained brown, and the treated cells showed the same distribution,but were stained more deeply. The average level of the control group andtreatment group were86.27±9.32and110.75±10.23individually, thedifference was statistically significant (P<0.05). Immunocytochemistry ofBcl-2: the cytoplasm of the control group was stained brown, and treatedcells showed the same distribution, but were stained less deeply. Theaverage level of the control group and treatment group were102.36±10.45and69.45±9.38individually, the difference was statistically significant(P<0.05).The mRNA expression of bax and p53increased significantly2hours after pulses, and bcl-2decreased2hours after pulses. The effect wasin a dose-dependent manner. Each treated group showed significantdifference in comparison to the control group (all P<0.05).
Conclusion: Imbalance of Bax and Bcl-2, p53were involved in theprocess of apoptosis induced by intense psPEF.
引文
[1] Bray F, Loos AH, Mc CP, et al. Trends in cervical squamous cell carcinomaincidence in13European countries: changing risk and the effects of screening[J].Cancer Epidemiology. Biomarkers and Prevention,2005,14(3):677-686
[2] Zarchi MK, Mousavi A, Malekzadeh M, et al. Conservative treatment in youngpatients with cervical cancer: a review[J]. Asian Pacific Journal of CancerPrevention,2010,11(3):589-594
[3] Bogden AE, Moreau JP, Eden PA. Proliferative response of human and animaltumors to surgical wounding of normal tissues: onset, duration and inhibition[J].British Journal of Cancer,1997,75(7):1021-1027
[4] Hofer OP, Shrayer D, Reichner JS. Wound-induced tumor progression[J]. Arch Surg,1998,133:383-389
[5] Weaver JC. Electroporation: a general phenomenon for manipulating cells andtissues[J]. Journal of Celluar Biochemisty,1993,51(4):426-435
[6] Okino M, Tomie H, Kanesada H, et al. Optimal electric conditions in electricalimpulse chemotherapy[J]. Japanese Journal of CancerResearch,1992,83(10):1095-1101
[7] Hofmann GA, Dev SB, Dimmer S, et al. Electroporation therapy: a new approachfor the treatment of head and neck cancer[J]. IEEE Transactions on BiomedicalEngineering,1999,(46)6:752-759
[8] Dev SB, Rabussay DP, Widera G, et al. Medical applications of electroporation[J].IEEE Transactions on Plasma Science,2000,28(1):206-223
[9] Mir LM, Julie Gehl, Gregor Sersa, et al. Standard operating procedures of theelectrochemotherapy: Instructions for the use of bleomycin or cisplatin administeredeither systemically or locally and electric pulses delivered by the CliniporatorTMbymeans of invasive or non-invasive electrodes[J]. EJC Supplements,2006,4:14-25
[10]Stacey M, Stickley J, Fox P, et al. Differential effects in cells exposed to ultra-short,high intensity electric fields: cell survival, DNA damage, and cell cycle analysis[J].Mutation Research,2003,542:65-75
[11]Sunao K, Naoyuki N, Hideto K, et al. Biological effects of narrow band pulsedelectric Fields[J]. IEEE Transactions on Dielectrics and ElectricalInsulation,2007,14(3):663-668
[12]Chen N, Garner AL, Chen G, et al. Nanosecond electric pulses penetrate the nucleusan enhance speckle formation[J]. Biochemical and Biophysical ResearchCommunications,2007,364:220-225
[13]Craviso GL, Chatterjee P, Maalouf G, et al. Nanosecond electric pulse-inducedincrease in intracellular calcium in adrenal chromaffin cells triggerscalcium-dependent catecholamine release[J]. Transactions on Dielectrics andElectrical Insulation,2009,16(5):1294-1301
[14]Joshi RP, Schoenbach KH. Bioelectric effects of intense ultrashort pulses[J].Critical Reviews in Biomedical Engineering,2010;38(3):255-304
[15]周玮，熊正爱，刘颖等.不可逆性电穿孔致HeLa细胞凋亡与坏死的作用研究[J].第三军医大学学报,2010,32(18):1941-1944
[16]刘晓云，熊正爱，李好山.白介素-21基因转导人宫颈癌细胞产生T细胞和NK细胞依赖的抗肿瘤作用[J].第三军医大学学报,2010,32(8):819-822
[17]杨晓燕，熊正爱，李成祥等.陡脉冲不可逆性电击穿治疗兔肝脏肿瘤研究[J].西南大学学报（自然科学版）,2009,31(8):120-125
[18]顾华妍，熊正爱，李成祥等.陡脉冲电场的生物学效应[J].中国组织工程研究与临床康复,2008,12(22):4201-4204
[19]Chenguo Yao, Dengbin Mo, Chengxiang Li, et al. Study of transmembranepotentials of inner and outer membranes induced by pulsed-electric-field model andsimulation. IEEE Transactions on Plasma Science [J],2007.35(5):1541-1549
[20]Chenguo Yao, Yan Mi, Chengxiang Li, et al. Study of transmembrane potentials oncellular inner and outer membrane--frequency response model and its filtercharacteristic simulation[J]. IEEE Transactions on Biomedical Engineering,2008,55(7):1792-1799.
[21]曾益新.肿瘤学.人民卫生出版社，1999
[22]彭黎明,王曾礼.细胞凋亡的基础与临床.人民卫生出版社，2000
[23]Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature.2001.411(6835):342-348
[24]Schoenbach KH, Shu Xiao, Ravindra P, et al. The effect of intense subnanosecondelectrical pulses on biological cells[J]. IEEE Transactions on PlasmaScience,2008,36(2):414-422
[25]Schoenbach KH, Katsuki S, Akiyama H, et al. Biological effects of intensesubnanosecond electrical pulses. Proceedings of the2006Power ModulatorConference, Arlington, VA,573-576
[26]Schoenbach KH, Xiao S, Camp JT. Subnanosecond electrical pulses for medicaltherapies and medical imaging. Expanded abstract, Proceedings of the2008PowerModulator Conference, Las Vegas, NV, p.60
[27]Camp JT, Xiao S, Beebe S, Peter F, et al. Bioelectric Studies with subnanosecondpulsed electric fields. digest of technical papers,2009IEEE Pulsed PowerConference, Washington, DC, June2009, p.876
[28]Baum CE, Stone AP and Tyo JS. Ultra-wideband, short-pulse electromagnetics8[M]. Springer Press.2007
[29]龙再全,姚陈果,李成祥,等.皮秒电场脉冲在肿瘤无创治疗中的聚焦特性[J].生物医学工程学杂志,2010,(5):1128-1132
[30]姚陈果,龙再全,孙才新,等.高强度皮秒电场脉冲无创治疗肿瘤天线设计[J].重庆大学学报,2010,33(7):29-34+57
[31]龙再全,姚陈果,孙才新,等. ps电场脉冲无创治疗肿瘤的方法及天线设计[J].高电压技术,2010,(5):1275-1280
[1] Chenguo Yao, Dengbin Mo, Chengxiang Li, et al. Study of transmembranepotentials of inner and outer membranes induced by pulsed-electric-field model andsimulation[J]. IEEE Transactions on Plasma Science,2007,35(5):1541-1549
[2] Chenguo Yao, Yan Mi, Chengxiang Li, et al. Study of transmembrane potentials oncellular inner and outer membrane--frequency response model and its filtercharacteristic simulation[J]. IEEE Transactions on Biomedical Engineering,2008,55(7):1792-1799.
[3] Ermolina I, Polevaya YL, Feldman YR, et al. Study of normal and malignant whiteblood cells by time domain dielectric spectroscopy[J]. IEEE Trans on Dielectr ElectrInsul,2001,8(2):253-261
[4] Feldman YR, Ermolina I, Hayashi Y. Time domain dielectric spectroscopy study ofbiological systems[J]. IEEE Trans on Dielectr Electr Insul,2003,10(5):728-753
[5] Schoenbach KH, Nuccitelli R, Beebe SJ. ZAP: Extreme Voltage could be aSurprisingly Delicate Tool in the Fight against Cancer[J]. IEEESpectrum,2006,43(8):20-26
[6] Weaver JC. Electroporation: a general phenomenon for manipulating cells andtissues[J]. Journal of Celluar Biochemisty,1993,51(4):426-435
[7] Okino M, Tomie H, Kanesada H, et al. Optimal electric conditions in electricalimpulse chemotherapy[J]. Japanese Journal of CancerResearch,1992,83(10):1095-1101
[8] Hofmann GA, Dev SB, Dimmer S, et al. Electroporation therapy: a new approachfor the treatment of head and neck cancer[J]. IEEE Transactions on BiomedicalEngineering,1999,(46)6:752-759
[9] Dev SB, Rabussay DP, Widera G, et al. Medical applications of electroporation[J].IEEE Transactions on Plasma Science,2000,28(1):206-223
[10]Rubinsky J, Onik G, Mikus P, et al. Optimal parameters for the destruction ofprostate cancer using irreversible electroporation[J]. J Urol,2008,180(6):2668-2674
[11]Stacey M, Stickley J, Fox P, et al. Differential effects in cells exposed to ultra-short,high intensity electric fields: cell survival, DNA damage, and cell cycle analysis[J].Mutation Research,2003,542:65-75
[12]Sunao K, Naoyuki N, Hideto K, et al. Biological effects of narrow band pulsedelectric Fields[J]. IEEE Transactions on Dielectrics and ElectricalInsulation,2007,14(3):663-668
[13]Chen N, Garner AL, Chen G, et al. Nanosecond electric pulses penetrate the nucleusan enhance speckle formation[J]. Biochemical and Biophysical ResearchCommunications,2007,364:220-225
[14]Craviso GL, Chatterjee P, Maalouf G, et al. Nanosecond electric pulse-inducedincrease in intracellular calcium in adrenal chromaffin cells triggerscalcium-dependent catecholamine release[J]. Transactions on Dielectrics andElectrical Insulation,2009,16(5):1294-1301
[15]Sersa G, Cemazar M, Parkins CS, et al. Tumour blood flow changes induced byapplication of electric pulses[J]. Eur J Cancer,1999,35(4):672-677
[16]Sersa G, Cemazar M, Miklavcic D, et al. Tumor blood flow modifying effect ofelectrochemotherapy with bleomycin[J]. Anticancer Res,1999,19(5B):4017-4022
[17]Nuccitelli R, Chen X, Pakhomov AG, et al. A new pulsed electric field therapy formelanoma disrupts the tumor's blood supply and causes complete remission withoutrecurrence[J]. Int J Cancer,2009,125(2):438-445
[18]Nuccitelli R, Pliquett U, Chen X, et al. Nanosecond pulsed electric fields causemelanomas to self-destruct[J]. Biochem Biophys ResCommun,2006,343(2):351-360
[19]韩旻.脉冲功率技术基础[M].北京：清华大学电气工程与应用电子技术系,2005
[20]周璧华，陈彬，石立华.电磁脉冲及其工程防护[M].北京：国防工业出版社,2003
[21]Schoenbach KH, Nuccitelli R, Beebe SJ. ZAP: Extreme Voltage could be aSurprisingly Delicate Tool in the Fight against Cancer[J]. IEEESpectrum,2006,43(8):20-26
[22]姚陈果，孙才新，米彦等.陡脉冲不可逆性电击穿治疗肿瘤的研究.高电压技术，2007，33(2):7-13
[23]赵东阳.一种生物医用皮秒级高压脉冲发生器的研制[D].重庆大学，2010
[24]姚陈果，赵东阳，米彦等.高压皮秒脉冲发生器的设计与实现[J].高电压技术,2010,36(7):1698-1703
[25]Bajracharya C, Xiao S, Baum CE, et al.Target detection with impulse radiatingantenna. IEEE Antennas and Wireless Propagation Letters,2011,(10):496-499
[26]Baum CE, Stone AP, Tyo JS. Ultra-Wideband, Short-Pulse Electromagnetics8[M].Springer Press,2007.
[27]曾益新.肿瘤学.人民卫生出版社，1999
[28]彭黎明,王曾礼.细胞凋亡的基础与临床.人民卫生出版社，2000
[29]Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature.2001.411(6835):342-348
[30]Schoenbach KH, Katsuki S, Akiyama H, et al. Biological effects of intensesubnanosecond electrical pulses. Proceedings of the2006Power ModulatorConference, Arlington, VA,573-576
[31]Schoenbach KH, Shu Xiao, Ravindra P, et al. The effect of intense subnanosecondelectrical pulses on biological cells. IEEE Transactions on PlasmaScience,2008,36(2):414-422
[32]Camp JT, Xiao S, Beebe S, Peter F, et al. Bioelectric Studies with subnanosecondpulsed electric fields. digest of technical papers,2009IEEE Pulsed PowerConference, Washington, DC, June2009, p.876-879
[1] Ravagnan L, Roumier T, Kroemer G. Mitochondria，the killer organelles and theirweapons[J]．J Cell Physiol,2002,192(2):131-137
[2]陶慧敏，王文文，关燕清.细胞程序式死亡途径的新进展[J].细胞生物学杂志，2008,30:563-568
[3] Orrenius S. Mitochondrial regulation of apoptotic cell death[J]. Toxicol Lett，2004，149(1-3):19-23.
[4] Armstrong JS. The role of the mitochondrial permeability transition in cell death[J].Mitochondrion,2006,6(5):225–234
[5] Joza N, Susin SA, Daugas E, et al. Essential role of the mitochondrialapoptosis-inducing factor in programmed cell death[J]. Nature,2001.410:549-554
[6] Slee EA, Harte MT, Kluck RM, et al. Ordering the cytochrome c-initiated caspasecascade: hierarchical activation of caspases-2,-3,-6,-7,-8, and-10in acaspase-9-dependent manner[J]. J Cell Biol,1999,144(2):281-292
[7] Stacey M, Stickley J, Fox P, et al. Differential effects in cells exposed to ultra-short,high intensity electric fields: cell survival, DNA damage, and cell cycle analysis.Mutat Res,2003,542(1-2):65-75
[8] Sunao K, Naoyuki N, Hideto K, et al. Biological effects of narrow band pulsedelectric fields. IEEE Trans on Dielectr Electr Insul,2007,14(3):663-668
[9] Chen N, Garner AL, Chen G, et al. Nanosecond electric pulses penetrate the nucleusan enhance speckle formation. Biochem and Biophy Res Commun,2007,364:220-225
[10]Craviso GL, Chatterjee P, Maalouf G, et al. Nanosecond electric pulse-inducedincrease in intracellular calcium in adrenal chromaffin cells triggerscalcium-dependent catecholamine release. Transactions on Dielectrics and ElectricalInsulation,2009,16(5):1294-1301
[11]Chen X, Chen X, Swanson RJ, et al. Histopathological follow-up by tissuemicro-array in a survival study after melanoma treated by nanosecond pulsedelectric fields (nsPEF). J Dermatolog Treat,2011,22(3):153-161.
[12]Chenguo Yao, Dengbin Mo, Chengxiang Li, et al. Study of transmembranepotentials of inner and outer membranes induced by pulsed-electric-field model andsimulation[J]. IEEE Transactions on Plasma Science,2007,35(5):1541-1549
[13]Chenguo Yao, Yan Mi, Chengxiang Li, et al. Study of transmembrane potentials oncellular inner and outer membrane--frequency response model and its filtercharacteristic simulation[J]. IEEE Transactions on Biomedical Engineering,2008,55(7):1792-1799.
[14]Ermolina I, Polevaya YL, Feldman YR, et al. Study of normal and malignant whiteblood cells by time domain dielectric spectroscopy[J]. IEEE Trans on Dielectr ElectrInsul,2001,8(2):253-261
[15]Feldman YR, Ermolina I, Hayashi Y. Time domain dielectric spectroscopy study ofbiological systems[J]. IEEE Trans on Dielectr Electr Insul,2003,10(5):728-753
[16]张向阳.细胞凋亡检测方法[J].济宁医学院学报,2008,31(3):259-260
[17]敖琳，曹佳.细胞凋亡中Ca2+的分子靶点[J]．癌变畸变突变，200l，13(3)：196-199
[18]Hajnóczky G, Csordás G, Das S, et al. Mitochondrial calcium signalling and celldeath: approaches for assessing the role of mitochondrial Ca2+uptake in apoptosis.Cell Calcium,2006,40(5-6):553-560.
[1] Gross A, McDonnell JM, Korsmeyer SJ. BCL-2family members and themitochondria in apoptosis. Genes Dev,1999,13(15):1899-1911.
[2] Burlacu A. Regulation of apoptosis by Bcl-2family proteins. J Cell Mol Med.2003,7(3):249-257.
[3] Hockenbery DM, Oltvai ZN, Yin XM, et al. Bcl-2functions in an antioxidantpathway to prevent apoptosis. Cell.1993,75(2):241-51.
[4] Lindsten T，Ross AJ，King A，et a1. The combined functions of proapoptotic Bcl-2family members bak and bax are essential for normal development of multipletissues[J]. Mol Cell，2000，6(6)：1389-1399.
[5]陶慧敏，王文文，关燕清.细胞程序式死亡途径的新进展[J].细胞生物学杂志，2008,30:563-568
[6] Wang X. The expanding role of mitochondira in apoptosis[J]．Genes Dev，2001，15(22)：2922-2933.
[7]宋今丹，杨恬，李丰.医学细胞生物学[M].人民卫生出版社，2008.
[8] Nuccitelli R, Chen X, Pakhomov AG, et a1. A new pulsed electric field therapy formelanoma disrupts the tumor's blood supply and causes complete remission withoutrecurrence[J]. Int J Cancer,2009,125(2):438-445.
[9] Ren W, Beebe SJ. An apoptosis targeted stimulus with nanosecond pulsed electricfields (nsPEFs) in E4squamous cell carcinoma. Apoptosis,2011,16(4):382-393.
[10]Hall EH, Schoenbach KH, Beebe SJ.Nanosecond pulsed electric fields induceapoptosis in p53-wildtype and p53-null HCT116colon carcinoma cells[J].Apoptosis,2007,12(9):1721-31.
[1] Schoenbach KH, Beebe SJ, Buescher ES. Intracellular effect of ultrashort electricalpulses[J].Bioelectromagnetics,2001,22(6):440-448
[2]姚陈果，陈新，李成祥等.脉冲电场作用下细胞频率特性仿真及窗口效应[J].生物医学工程学杂志,2011.28(1):12-17
[3] Esser AT, Smith KC, Gowrishankar T.R, et al. Mechanisms for the IntracellularManipulation of Organelles by Conventional Electroporation[J].BiophysJ,2010,98(11):2506-2514
[4] Smith KC, Weaver JC.Active Mechanisms Are Needed to Describe Cell Responsesto Submicrosecond, Megavolt-per-Meter Pulses: Cell Models for UltrashortPulses[J].Biophysical Journal,2008,95(4):1547-1563
[5] Gowrishankar TR, Weaver JC. Electrical behavior and pore accumulation in amulticellular model for conventional andsupra-electroporation[J].Biochem.Biophys.Res.Commun.2006.349(2):643-653.
[6] Nesin OM, Pakhomova ON, Xiao S, et al.Manipulation of cell volume andmembrane pore comparison following single cell permeabilization with60-and600-ns electric pulses[J].Biochim Biophys Acta.1808(3):792-801
[7] Batista Napotnik T, Wu YH, Gundersen MA, et al.Nanosecond electric pulses causemitochondrial membrane permeabilization in Jurkat cells.Bioelectromagnetics,2012,33(3):257-264
[8] Esser AT, Smith KC, Gowrishankar TR, et al. Towards solid tumor treatment bynanosecond pulsed electric felds[J].Technol.Cancer Res.Treat.2009.8(4):289-306.
[9] Smith KC, Weaver JC. Transmembrane molecular transport during versus afterextremely large, nanosecond electric pulses[J].Biochem Biophys ResCommun.412(1):8-12
[10]Vernier PT, Sun Y, Marcu L, et al. Calcium bursts induced by nanosecond electricpulses[J]. Biochem Biophys Res Commun.2003,310(2):286-295.
[11]唐均英,吴晓娟,姚陈果,姚珍薇,孙才新.纳秒级陡脉冲对人卵巢癌细胞SKOV3凋亡及细胞内Ca2+浓度的影响[J].现代妇产科进展.2007.l6(11):827-831
[12]杨方黎,唐均英,米彦,姚陈果,王剑飞.纳秒级陡脉冲电场诱导人黑色素瘤细胞裸鼠皮下移植瘤凋亡作用及机制[J].第四军医大学学报.2009.30(13):1227-1230
[13]Weaver JC. Electroporation of biological membranes from multicellular tonanoscales[J]. IEEE Trans Dielect Elect Insul.2003;10(5):754-768.
[14]Hall EH, Schoenbach KH,Beebe SJ. Nanosecond pulsed electric fields induceapoptosis in p53-wildtype and p53-null HCT116colon carcinomacells[J].Apoptosis.2007.12(9):1721-1731.
[15]Ren W, Beebe SJ.An apoptosis targeted stimulus with nanosecond pulsed electricfields(nsPEFs) in E4squamous cell carcinoma[J].Apoptosis.2011.16(4):382-393.
[16]Wartenberg M, Wirtz N, Grob A, et al. Direct current electrical fields induceapoptosis in oral mucosa cancer cells by NADPH oxidase-derived reactive oxygenspecies[J]. Bioelectromagnetics.2008;29(1):47-54.
[17]Goldmann WH. Actin: A molecular wire, an electrical cable[J]? Cell BiolInt,2008,32(7):869-870.
[18]Kanthou C, Kranjc S, Sersa G, et al. The endothelial cytoskeleton as a target ofelectroporation-based therapies[J]. Mol Cancer Ther,2006,5(12):3145-3152.
[19]Xiao D, Tang L, Zeng C, et al.Effect of actin cytoskeleton disruption on electricpulse-induced apoptosis and electroporation in tumour cells[J].CellBiol.Int.2011.35(2):99-104
[20]Walker K3rd, Pakhomova ON, Kolb J, et al. Oxygen enhances lethal effect ofhigh-intensity, ultrashort electrical pulses[J]. Bioelectromagnetics[J].2006;27(3):221-225.
[21]Joshi RP, Schoenbach KH.Bioelectric Effects of Intense Ultrashort Pulses[J].CritRev Biomed Eng,2010.38(3):255-304
[2] Zarchi MK, Mousavi A, Malekzadeh M, et al. Conservative treatment in youngpatients with cervical cancer: a review[J]. Asian Pacific Journal of CancerPrevention,2010,11(3):589-594
[3] Bogden AE, Moreau JP, Eden PA. Proliferative response of human and animaltumors to surgical wounding of normal tissues: onset, duration and inhibition[J].British Journal of Cancer,1997,75(7):1021-1027
[4] Hofer OP, Shrayer D, Reichner JS. Wound-induced tumor progression[J]. Arch Surg,1998,133:383-389
[5] Weaver JC. Electroporation: a general phenomenon for manipulating cells andtissues[J]. Journal of Celluar Biochemisty,1993,51(4):426-435
[6] Okino M, Tomie H, Kanesada H, et al. Optimal electric conditions in electricalimpulse chemotherapy[J]. Japanese Journal of CancerResearch,1992,83(10):1095-1101
[7] Hofmann GA, Dev SB, Dimmer S, et al. Electroporation therapy: a new approachfor the treatment of head and neck cancer[J]. IEEE Transactions on BiomedicalEngineering,1999,(46)6:752-759
[8] Dev SB, Rabussay DP, Widera G, et al. Medical applications of electroporation[J].IEEE Transactions on Plasma Science,2000,28(1):206-223
[9] Mir LM, Julie Gehl, Gregor Sersa, et al. Standard operating procedures of theelectrochemotherapy: Instructions for the use of bleomycin or cisplatin administeredeither systemically or locally and electric pulses delivered by the CliniporatorTMbymeans of invasive or non-invasive electrodes[J]. EJC Supplements,2006,4:14-25
[10]Stacey M, Stickley J, Fox P, et al. Differential effects in cells exposed to ultra-short,high intensity electric fields: cell survival, DNA damage, and cell cycle analysis[J].Mutation Research,2003,542:65-75
[11]Sunao K, Naoyuki N, Hideto K, et al. Biological effects of narrow band pulsedelectric Fields[J]. IEEE Transactions on Dielectrics and ElectricalInsulation,2007,14(3):663-668
[12]Chen N, Garner AL, Chen G, et al. Nanosecond electric pulses penetrate the nucleusan enhance speckle formation[J]. Biochemical and Biophysical ResearchCommunications,2007,364:220-225
[13]Craviso GL, Chatterjee P, Maalouf G, et al. Nanosecond electric pulse-inducedincrease in intracellular calcium in adrenal chromaffin cells triggerscalcium-dependent catecholamine release[J]. Transactions on Dielectrics andElectrical Insulation,2009,16(5):1294-1301
[14]Joshi RP, Schoenbach KH. Bioelectric effects of intense ultrashort pulses[J].Critical Reviews in Biomedical Engineering,2010;38(3):255-304
[15]周玮，熊正爱，刘颖等.不可逆性电穿孔致HeLa细胞凋亡与坏死的作用研究[J].第三军医大学学报,2010,32(18):1941-1944
[16]刘晓云，熊正爱，李好山.白介素-21基因转导人宫颈癌细胞产生T细胞和NK细胞依赖的抗肿瘤作用[J].第三军医大学学报,2010,32(8):819-822
[17]杨晓燕，熊正爱，李成祥等.陡脉冲不可逆性电击穿治疗兔肝脏肿瘤研究[J].西南大学学报（自然科学版）,2009,31(8):120-125
[18]顾华妍，熊正爱，李成祥等.陡脉冲电场的生物学效应[J].中国组织工程研究与临床康复,2008,12(22):4201-4204
[19]Chenguo Yao, Dengbin Mo, Chengxiang Li, et al. Study of transmembranepotentials of inner and outer membranes induced by pulsed-electric-field model andsimulation. IEEE Transactions on Plasma Science [J],2007.35(5):1541-1549
[20]Chenguo Yao, Yan Mi, Chengxiang Li, et al. Study of transmembrane potentials oncellular inner and outer membrane--frequency response model and its filtercharacteristic simulation[J]. IEEE Transactions on Biomedical Engineering,2008,55(7):1792-1799.
[21]曾益新.肿瘤学.人民卫生出版社，1999
[22]彭黎明,王曾礼.细胞凋亡的基础与临床.人民卫生出版社，2000
[23]Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature.2001.411(6835):342-348
[24]Schoenbach KH, Shu Xiao, Ravindra P, et al. The effect of intense subnanosecondelectrical pulses on biological cells[J]. IEEE Transactions on PlasmaScience,2008,36(2):414-422
[25]Schoenbach KH, Katsuki S, Akiyama H, et al. Biological effects of intensesubnanosecond electrical pulses. Proceedings of the2006Power ModulatorConference, Arlington, VA,573-576
[26]Schoenbach KH, Xiao S, Camp JT. Subnanosecond electrical pulses for medicaltherapies and medical imaging. Expanded abstract, Proceedings of the2008PowerModulator Conference, Las Vegas, NV, p.60
[27]Camp JT, Xiao S, Beebe S, Peter F, et al. Bioelectric Studies with subnanosecondpulsed electric fields. digest of technical papers,2009IEEE Pulsed PowerConference, Washington, DC, June2009, p.876
[28]Baum CE, Stone AP and Tyo JS. Ultra-wideband, short-pulse electromagnetics8[M]. Springer Press.2007
[29]龙再全,姚陈果,李成祥,等.皮秒电场脉冲在肿瘤无创治疗中的聚焦特性[J].生物医学工程学杂志,2010,(5):1128-1132
[30]姚陈果,龙再全,孙才新,等.高强度皮秒电场脉冲无创治疗肿瘤天线设计[J].重庆大学学报,2010,33(7):29-34+57
[31]龙再全,姚陈果,孙才新,等. ps电场脉冲无创治疗肿瘤的方法及天线设计[J].高电压技术,2010,(5):1275-1280
[1] Chenguo Yao, Dengbin Mo, Chengxiang Li, et al. Study of transmembranepotentials of inner and outer membranes induced by pulsed-electric-field model andsimulation[J]. IEEE Transactions on Plasma Science,2007,35(5):1541-1549
[2] Chenguo Yao, Yan Mi, Chengxiang Li, et al. Study of transmembrane potentials oncellular inner and outer membrane--frequency response model and its filtercharacteristic simulation[J]. IEEE Transactions on Biomedical Engineering,2008,55(7):1792-1799.
[3] Ermolina I, Polevaya YL, Feldman YR, et al. Study of normal and malignant whiteblood cells by time domain dielectric spectroscopy[J]. IEEE Trans on Dielectr ElectrInsul,2001,8(2):253-261
[4] Feldman YR, Ermolina I, Hayashi Y. Time domain dielectric spectroscopy study ofbiological systems[J]. IEEE Trans on Dielectr Electr Insul,2003,10(5):728-753
[5] Schoenbach KH, Nuccitelli R, Beebe SJ. ZAP: Extreme Voltage could be aSurprisingly Delicate Tool in the Fight against Cancer[J]. IEEESpectrum,2006,43(8):20-26
[6] Weaver JC. Electroporation: a general phenomenon for manipulating cells andtissues[J]. Journal of Celluar Biochemisty,1993,51(4):426-435
[7] Okino M, Tomie H, Kanesada H, et al. Optimal electric conditions in electricalimpulse chemotherapy[J]. Japanese Journal of CancerResearch,1992,83(10):1095-1101
[8] Hofmann GA, Dev SB, Dimmer S, et al. Electroporation therapy: a new approachfor the treatment of head and neck cancer[J]. IEEE Transactions on BiomedicalEngineering,1999,(46)6:752-759
[9] Dev SB, Rabussay DP, Widera G, et al. Medical applications of electroporation[J].IEEE Transactions on Plasma Science,2000,28(1):206-223
[10]Rubinsky J, Onik G, Mikus P, et al. Optimal parameters for the destruction ofprostate cancer using irreversible electroporation[J]. J Urol,2008,180(6):2668-2674
[11]Stacey M, Stickley J, Fox P, et al. Differential effects in cells exposed to ultra-short,high intensity electric fields: cell survival, DNA damage, and cell cycle analysis[J].Mutation Research,2003,542:65-75
[12]Sunao K, Naoyuki N, Hideto K, et al. Biological effects of narrow band pulsedelectric Fields[J]. IEEE Transactions on Dielectrics and ElectricalInsulation,2007,14(3):663-668
[13]Chen N, Garner AL, Chen G, et al. Nanosecond electric pulses penetrate the nucleusan enhance speckle formation[J]. Biochemical and Biophysical ResearchCommunications,2007,364:220-225
[14]Craviso GL, Chatterjee P, Maalouf G, et al. Nanosecond electric pulse-inducedincrease in intracellular calcium in adrenal chromaffin cells triggerscalcium-dependent catecholamine release[J]. Transactions on Dielectrics andElectrical Insulation,2009,16(5):1294-1301
[15]Sersa G, Cemazar M, Parkins CS, et al. Tumour blood flow changes induced byapplication of electric pulses[J]. Eur J Cancer,1999,35(4):672-677
[16]Sersa G, Cemazar M, Miklavcic D, et al. Tumor blood flow modifying effect ofelectrochemotherapy with bleomycin[J]. Anticancer Res,1999,19(5B):4017-4022
[17]Nuccitelli R, Chen X, Pakhomov AG, et al. A new pulsed electric field therapy formelanoma disrupts the tumor's blood supply and causes complete remission withoutrecurrence[J]. Int J Cancer,2009,125(2):438-445
[18]Nuccitelli R, Pliquett U, Chen X, et al. Nanosecond pulsed electric fields causemelanomas to self-destruct[J]. Biochem Biophys ResCommun,2006,343(2):351-360
[19]韩旻.脉冲功率技术基础[M].北京：清华大学电气工程与应用电子技术系,2005
[20]周璧华，陈彬，石立华.电磁脉冲及其工程防护[M].北京：国防工业出版社,2003
[21]Schoenbach KH, Nuccitelli R, Beebe SJ. ZAP: Extreme Voltage could be aSurprisingly Delicate Tool in the Fight against Cancer[J]. IEEESpectrum,2006,43(8):20-26
[22]姚陈果，孙才新，米彦等.陡脉冲不可逆性电击穿治疗肿瘤的研究.高电压技术，2007，33(2):7-13
[23]赵东阳.一种生物医用皮秒级高压脉冲发生器的研制[D].重庆大学，2010
[24]姚陈果，赵东阳，米彦等.高压皮秒脉冲发生器的设计与实现[J].高电压技术,2010,36(7):1698-1703
[25]Bajracharya C, Xiao S, Baum CE, et al.Target detection with impulse radiatingantenna. IEEE Antennas and Wireless Propagation Letters,2011,(10):496-499
[26]Baum CE, Stone AP, Tyo JS. Ultra-Wideband, Short-Pulse Electromagnetics8[M].Springer Press,2007.
[27]曾益新.肿瘤学.人民卫生出版社，1999
[28]彭黎明,王曾礼.细胞凋亡的基础与临床.人民卫生出版社，2000
[29]Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature.2001.411(6835):342-348
[30]Schoenbach KH, Katsuki S, Akiyama H, et al. Biological effects of intensesubnanosecond electrical pulses. Proceedings of the2006Power ModulatorConference, Arlington, VA,573-576
[31]Schoenbach KH, Shu Xiao, Ravindra P, et al. The effect of intense subnanosecondelectrical pulses on biological cells. IEEE Transactions on PlasmaScience,2008,36(2):414-422
[32]Camp JT, Xiao S, Beebe S, Peter F, et al. Bioelectric Studies with subnanosecondpulsed electric fields. digest of technical papers,2009IEEE Pulsed PowerConference, Washington, DC, June2009, p.876-879
[1] Ravagnan L, Roumier T, Kroemer G. Mitochondria，the killer organelles and theirweapons[J]．J Cell Physiol,2002,192(2):131-137
[2]陶慧敏，王文文，关燕清.细胞程序式死亡途径的新进展[J].细胞生物学杂志，2008,30:563-568
[3] Orrenius S. Mitochondrial regulation of apoptotic cell death[J]. Toxicol Lett，2004，149(1-3):19-23.
[4] Armstrong JS. The role of the mitochondrial permeability transition in cell death[J].Mitochondrion,2006,6(5):225–234
[5] Joza N, Susin SA, Daugas E, et al. Essential role of the mitochondrialapoptosis-inducing factor in programmed cell death[J]. Nature,2001.410:549-554
[6] Slee EA, Harte MT, Kluck RM, et al. Ordering the cytochrome c-initiated caspasecascade: hierarchical activation of caspases-2,-3,-6,-7,-8, and-10in acaspase-9-dependent manner[J]. J Cell Biol,1999,144(2):281-292
[7] Stacey M, Stickley J, Fox P, et al. Differential effects in cells exposed to ultra-short,high intensity electric fields: cell survival, DNA damage, and cell cycle analysis.Mutat Res,2003,542(1-2):65-75
[8] Sunao K, Naoyuki N, Hideto K, et al. Biological effects of narrow band pulsedelectric fields. IEEE Trans on Dielectr Electr Insul,2007,14(3):663-668
[9] Chen N, Garner AL, Chen G, et al. Nanosecond electric pulses penetrate the nucleusan enhance speckle formation. Biochem and Biophy Res Commun,2007,364:220-225
[10]Craviso GL, Chatterjee P, Maalouf G, et al. Nanosecond electric pulse-inducedincrease in intracellular calcium in adrenal chromaffin cells triggerscalcium-dependent catecholamine release. Transactions on Dielectrics and ElectricalInsulation,2009,16(5):1294-1301
[11]Chen X, Chen X, Swanson RJ, et al. Histopathological follow-up by tissuemicro-array in a survival study after melanoma treated by nanosecond pulsedelectric fields (nsPEF). J Dermatolog Treat,2011,22(3):153-161.
[12]Chenguo Yao, Dengbin Mo, Chengxiang Li, et al. Study of transmembranepotentials of inner and outer membranes induced by pulsed-electric-field model andsimulation[J]. IEEE Transactions on Plasma Science,2007,35(5):1541-1549
[13]Chenguo Yao, Yan Mi, Chengxiang Li, et al. Study of transmembrane potentials oncellular inner and outer membrane--frequency response model and its filtercharacteristic simulation[J]. IEEE Transactions on Biomedical Engineering,2008,55(7):1792-1799.
[14]Ermolina I, Polevaya YL, Feldman YR, et al. Study of normal and malignant whiteblood cells by time domain dielectric spectroscopy[J]. IEEE Trans on Dielectr ElectrInsul,2001,8(2):253-261
[15]Feldman YR, Ermolina I, Hayashi Y. Time domain dielectric spectroscopy study ofbiological systems[J]. IEEE Trans on Dielectr Electr Insul,2003,10(5):728-753
[16]张向阳.细胞凋亡检测方法[J].济宁医学院学报,2008,31(3):259-260
[17]敖琳，曹佳.细胞凋亡中Ca2+的分子靶点[J]．癌变畸变突变，200l，13(3)：196-199
[18]Hajnóczky G, Csordás G, Das S, et al. Mitochondrial calcium signalling and celldeath: approaches for assessing the role of mitochondrial Ca2+uptake in apoptosis.Cell Calcium,2006,40(5-6):553-560.
[1] Gross A, McDonnell JM, Korsmeyer SJ. BCL-2family members and themitochondria in apoptosis. Genes Dev,1999,13(15):1899-1911.
[2] Burlacu A. Regulation of apoptosis by Bcl-2family proteins. J Cell Mol Med.2003,7(3):249-257.
[3] Hockenbery DM, Oltvai ZN, Yin XM, et al. Bcl-2functions in an antioxidantpathway to prevent apoptosis. Cell.1993,75(2):241-51.
[4] Lindsten T，Ross AJ，King A，et a1. The combined functions of proapoptotic Bcl-2family members bak and bax are essential for normal development of multipletissues[J]. Mol Cell，2000，6(6)：1389-1399.
[5]陶慧敏，王文文，关燕清.细胞程序式死亡途径的新进展[J].细胞生物学杂志，2008,30:563-568
[6] Wang X. The expanding role of mitochondira in apoptosis[J]．Genes Dev，2001，15(22)：2922-2933.
[7]宋今丹，杨恬，李丰.医学细胞生物学[M].人民卫生出版社，2008.
[8] Nuccitelli R, Chen X, Pakhomov AG, et a1. A new pulsed electric field therapy formelanoma disrupts the tumor's blood supply and causes complete remission withoutrecurrence[J]. Int J Cancer,2009,125(2):438-445.
[9] Ren W, Beebe SJ. An apoptosis targeted stimulus with nanosecond pulsed electricfields (nsPEFs) in E4squamous cell carcinoma. Apoptosis,2011,16(4):382-393.
[10]Hall EH, Schoenbach KH, Beebe SJ.Nanosecond pulsed electric fields induceapoptosis in p53-wildtype and p53-null HCT116colon carcinoma cells[J].Apoptosis,2007,12(9):1721-31.
[1] Schoenbach KH, Beebe SJ, Buescher ES. Intracellular effect of ultrashort electricalpulses[J].Bioelectromagnetics,2001,22(6):440-448
[2]姚陈果，陈新，李成祥等.脉冲电场作用下细胞频率特性仿真及窗口效应[J].生物医学工程学杂志,2011.28(1):12-17
[3] Esser AT, Smith KC, Gowrishankar T.R, et al. Mechanisms for the IntracellularManipulation of Organelles by Conventional Electroporation[J].BiophysJ,2010,98(11):2506-2514
[4] Smith KC, Weaver JC.Active Mechanisms Are Needed to Describe Cell Responsesto Submicrosecond, Megavolt-per-Meter Pulses: Cell Models for UltrashortPulses[J].Biophysical Journal,2008,95(4):1547-1563
[5] Gowrishankar TR, Weaver JC. Electrical behavior and pore accumulation in amulticellular model for conventional andsupra-electroporation[J].Biochem.Biophys.Res.Commun.2006.349(2):643-653.
[6] Nesin OM, Pakhomova ON, Xiao S, et al.Manipulation of cell volume andmembrane pore comparison following single cell permeabilization with60-and600-ns electric pulses[J].Biochim Biophys Acta.1808(3):792-801
[7] Batista Napotnik T, Wu YH, Gundersen MA, et al.Nanosecond electric pulses causemitochondrial membrane permeabilization in Jurkat cells.Bioelectromagnetics,2012,33(3):257-264
[8] Esser AT, Smith KC, Gowrishankar TR, et al. Towards solid tumor treatment bynanosecond pulsed electric felds[J].Technol.Cancer Res.Treat.2009.8(4):289-306.
[9] Smith KC, Weaver JC. Transmembrane molecular transport during versus afterextremely large, nanosecond electric pulses[J].Biochem Biophys ResCommun.412(1):8-12
[10]Vernier PT, Sun Y, Marcu L, et al. Calcium bursts induced by nanosecond electricpulses[J]. Biochem Biophys Res Commun.2003,310(2):286-295.
[11]唐均英,吴晓娟,姚陈果,姚珍薇,孙才新.纳秒级陡脉冲对人卵巢癌细胞SKOV3凋亡及细胞内Ca2+浓度的影响[J].现代妇产科进展.2007.l6(11):827-831
[12]杨方黎,唐均英,米彦,姚陈果,王剑飞.纳秒级陡脉冲电场诱导人黑色素瘤细胞裸鼠皮下移植瘤凋亡作用及机制[J].第四军医大学学报.2009.30(13):1227-1230
[13]Weaver JC. Electroporation of biological membranes from multicellular tonanoscales[J]. IEEE Trans Dielect Elect Insul.2003;10(5):754-768.
[14]Hall EH, Schoenbach KH,Beebe SJ. Nanosecond pulsed electric fields induceapoptosis in p53-wildtype and p53-null HCT116colon carcinomacells[J].Apoptosis.2007.12(9):1721-1731.
[15]Ren W, Beebe SJ.An apoptosis targeted stimulus with nanosecond pulsed electricfields(nsPEFs) in E4squamous cell carcinoma[J].Apoptosis.2011.16(4):382-393.
[16]Wartenberg M, Wirtz N, Grob A, et al. Direct current electrical fields induceapoptosis in oral mucosa cancer cells by NADPH oxidase-derived reactive oxygenspecies[J]. Bioelectromagnetics.2008;29(1):47-54.
[17]Goldmann WH. Actin: A molecular wire, an electrical cable[J]? Cell BiolInt,2008,32(7):869-870.
[18]Kanthou C, Kranjc S, Sersa G, et al. The endothelial cytoskeleton as a target ofelectroporation-based therapies[J]. Mol Cancer Ther,2006,5(12):3145-3152.
[19]Xiao D, Tang L, Zeng C, et al.Effect of actin cytoskeleton disruption on electricpulse-induced apoptosis and electroporation in tumour cells[J].CellBiol.Int.2011.35(2):99-104
[20]Walker K3rd, Pakhomova ON, Kolb J, et al. Oxygen enhances lethal effect ofhigh-intensity, ultrashort electrical pulses[J]. Bioelectromagnetics[J].2006;27(3):221-225.
[21]Joshi RP, Schoenbach KH.Bioelectric Effects of Intense Ultrashort Pulses[J].CritRev Biomed Eng,2010.38(3):255-304