【作者】 华媛媛；

【导师】 熊正爱；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2012， 博士

【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Ca²⁺的浓度[Ca²⁺]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Ca²⁺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Ca²⁺浓度[Ca²⁺]i升高，呈现剂量依赖性，随脉冲个数增加，Ca²⁺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。更多还原

【Abstract】 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.更多还原