【作者】 吴晓娟；

【导师】 姚珍薇；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2008， 硕士

【摘要】 脉冲电场因其独特的电生物学效应,已在肿瘤治疗中显示出良好的应用前景。进一步研究发现纳秒级脉冲电场（nanosecond pulsed electric fields ,nsPEF）作用下细胞出现了一系列与微秒级脉冲电场诱导细胞膜电击穿现象截然不同的反应,即细胞内电处理效应。纳秒级脉冲由于其等值频率很高能穿透细胞膜（外膜）,使得包括细胞核、线粒体、内质网膜等（内膜）和细胞信号转导等出现一系列的功能性改变,为诱导肿瘤细胞发生凋亡提供了可能。近年来,纳秒级脉冲电场诱导肿瘤细胞凋亡相关实验逐渐成为国内外电脉冲生物应用领域研究焦点。但目前对纳秒级脉冲电场治疗肿瘤的凋亡形态学报道较少;研究对象主要为白血病和结肠癌,病种较局限;凋亡机制尚未明确。因此本实验拟通过对纳秒级陡脉冲（陡脉冲与纳秒级脉冲相结合的电场）体外诱导人卵巢癌细胞凋亡的形态及功能学观察及凋亡机制探讨,为纳秒级陡脉冲应用于卵巢癌治疗提供理论及实验依据。第一部分纳秒级陡脉冲对人卵巢癌细胞的生长抑制和凋亡诱导作用目的:探讨纳秒级陡脉冲对人卵巢癌细胞的生长抑制和凋亡诱导作用。方法:采用人卵巢癌细胞SKOV3,用电场强度为8.5（低）,10（中）,12.5（高）kV/cm的纳秒级陡脉冲分别作用于SKOV3细胞5min。应用MTT比色法,倒置显微镜,荧光显微镜及Annexin-FITC技术等方法进行检测和观察。结果:中（10 kV/cm）、高（12.5 kV/cm）场强处理组对SKOV3细胞活性具有明显抑制作用,呈剂量依赖性,在处理后2～4h内呈时间依赖性;倒置显微镜和荧光显微镜下观察到典型的凋亡细胞形态;流式细胞仪示,中、高场强处理组早期凋亡率分别为（22.21±2.71）%、（57.30±6.51）%,与对照组（3.04±0.44）%比较均有显著性差异。结论:一定场强及时间范围内的纳秒级陡脉冲能明显抑制SKOV3细胞增殖,亦诱导SKOV3细胞凋亡。第二部分纳秒级陡脉冲诱导人卵巢癌细胞凋亡的电镜观察及凋亡机制的研究目的:观察纳秒级陡脉冲诱导人卵巢癌细胞SKOV3凋亡的超微结构变化及其对细胞内钙离子浓度即[Ca²⁺]i的影响。方法:纳秒级陡脉冲场强为10kV/cm,脉宽为100ns,频率为1Hz,作用5min。SKOV3细胞经纳秒级陡脉冲作用后4h,采用透射电子显微镜、扫描电子显微镜检测细胞凋亡;以Fluo-3/AM为细胞内钙离子的荧光指示剂,用激光扫描共聚焦显微镜检测纳秒级陡脉冲对SKOV3细胞[Ca²⁺]i的影响及[Ca²⁺]i变化时的Ca²⁺来源。结果:透射及扫描电子显微镜观察到典型的凋亡细胞形态。纳秒级陡脉冲可明显升高细胞[Ca²⁺]i（P<0.01）,而与细胞外钙离子浓度无关（P>0.05）。结论:纳秒级陡脉冲能够诱导SKOV3细胞凋亡。细胞内钙释放引起的钙离子升高,可能是纳秒级陡脉冲诱导肿瘤细胞凋亡的机制之一。更多还原

【Abstract】 The pulsed electric fields （PEF）,due to its unique electrobiological effect,has showed good prospective application for tumor therapy.Futher research found that Intracellular changes very different from electroporation phenomenon could be induced by nanosecond pulsed electric fields （nsPEF）.Compared with the traditional electroporation,this phenomenon is termed intracellular electromanipulation （IEM）.nsPEF can penetrate cell interior due to its high equivalent frequency, leading to a series of functional changes in nucleolus, mitochondria,endoplasmic reticulum,and cell signal transfer,which provided possible foundation of apoptosis under nsPEF.Nowadays,experiment and mechanism research on cancer cell apoptosis induced by nsPEF have become a research hotspot in the field of tumor therapy internationally.But in these articles reserch on the apoptotic morphological feature under nsPEF was less,and disease entities were limited.in addition,the exact mechanism of apoptosis induced by nsPEF remains to be studied furtherly.So in this study,we plan to investigate the apoptotic morphological and functional changes,in order to ascertain the mechanism of apoptosis induced by nanosecond steep pulse on human ovarian carcinoma cell Line SKOV3 in vitro.We hope to provide a theoretical and experimental basement for the clinical application of nanosecond steep pulse therapy. PART I:GROWTH-INHIBITED AND APOPTOSIS-INDUCED EFFECTS OF NANOSECOND STEEP PULSE ON HUMAN OVARIAN CARCINOMA CELL LINE SKOV3Objective:To investigate the effect of Nanosecond Steep Pulse on growth and apoptosis in human ovarian carcinoma cells in vitro.Methods:After SKOV3 cells were treated with nanosecond steep pulse of 8.5,10,12.5 kilovolts per centimeter（kV/cm） amplitude respectively,MTT assay,invert microscopy,fluorescence microscopy and AnnexinV-FITC were performed to indentify the inhibitory effect of nanosecond steep pulse on cell proliferation and the induction of apoptosis in human ovarian carcinoma cell Line SKOV3.Results:Nanosecond steep pulse with 10 and 12.5 kV/cm significantly inhibited the viability of SKOV3 cells in a dose-dependent manner,and in a time-dependent manner within the range of 2 h to 4 h.The typical changes of SKOV3 cell apoptosis were observed by invert microscope and fluorescence microscopy.Early apoptotic rates of SKOV3 treated with 10,12.5 kV/cm nanosecond steep pulse were（22.21±2.71）% and（57.30±6.51）%, respectively,significantly higher than those of the control group（3.04±0.44）% （P<0.05）.Conclusion:Nanosecond steep pulse in a certain range can inhibite the growth of SKOV3 cells as well as induce the apoptosis. PART II:ELECTRON MICROSCOPIC OBSERVATION AND MECHANISMS STUDIES ON APOPTOSIS OF HUMAN OVARIAN CARCINOMA CELL LINE SKOV3 INDUCED BY NANOSECOND STEEP PULSEObjective:To investigate the apoptotic morphological features and changes of the intracellular calcium concentration ([Ca²⁺]i ) induced by nanosecond steep pulse on human ovarian carcinoma cell Line SKOV3 in vitro.Methods:SKOV3 cells were exposed to nanosecond steep pulse （10kV/cm,100ns,1 Hz in 5 minutes）. At 4h after treatment, the apoptosis of SKOV3 cells was detected by transmission electron microscope and scanning electron microscope.The change of [Ca²⁺]i in SKOV3 cells and the source of Ca²⁺ during [Ca²⁺]i change were observed by laser scanning confocal microscope using Fluo-3/AM as the calcium fluorenscent indicator.Results:The typical morphological features of apoptotic cells were observed by transmission electron microscope and scanning electron microscope.The [Ca²⁺]i was markedly increased by treatment with nanosecond steep pulse（P<0.01）,while the increase showed no relationship to the extracellular calcium concentration（P>0.05）. Conclusion:Nanosecond steep pulse can induce apoptosis of SKOV3 cells.The increase of [Ca²⁺]i through release of intracellular calcium might be one of the mechanisms of the apoptosis.更多还原