【作者】 米彦；

【导师】 孙才新；

【作者基本信息】 重庆大学 ， 电气工程， 2009， 博士

【摘要】 恶性肿瘤严重威胁人类生命健康,传统的治疗手段由于受其适应症、禁忌症和副作用等因素的限制,对恶性肿瘤的疗效仍不够理想。传统肿瘤治疗方法的机理在于杀伤肿瘤细胞并抑制其生长与增殖,因而只能延缓肿瘤的进展,不能达到治愈的目的。而诱导肿瘤细胞凋亡能使肿瘤组织缩小甚至消失,并减少对正常细胞的损害和治疗过程中的不良反应,因此,研究诱导肿瘤细胞凋亡的方法与机制,对于肿瘤治疗有着特别重要的意义。为此,本文在传统电疗法的基础上,通过阐述各种参数脉冲电场的生物电效应共性基础理论,深入研究纳秒脉冲电场诱导肿瘤凋亡的方法和机制,取得的主要成果有:①分别建立了球形细胞的多层介电模型、等效电路模型和场-路复合模型,提出了各种模型内外膜跨膜电位的计算方法,分析了内外膜跨膜电位对全时/频段(微秒-纳秒-皮秒)范围内脉冲电场的响应规律,从而提出了脉冲电场的脉宽-场强脉冲参数窗口效应和电导率-介电常数细胞电参数窗口效应。②以传统脉冲功率技术中的电容充放电为基本原理,结合高压大功率电力电子开关及其触发技术、基于数字电位器和线性光耦的程控调压技术,研制出了一套高场强纳秒脉冲肿瘤治疗仪样机,该样机稳定可靠,并能对输出指数衰减纳秒脉冲的电压峰值(0～25kV)、脉冲宽度(100ns～1μs)、重复频率(1～100Hz)和脉冲个数等参数进行独立、精确控制。③以离体人浆液囊腺性卵巢癌SKOV3肿瘤细胞为对象,采用场强10kV/cm、脉宽100ns的高场强纳秒脉冲电场进行处理,通过流式细胞术检测、扫描电子显微镜观察、透射电子显微镜观察、AO(吖啶橙)/EB(溴化乙啶)荧光检测、细胞内钙离子浓度检测等手段,证实了纳秒脉冲电场能有效诱导离体肿瘤细胞凋亡,其机制是通过激活细胞内钙库(内质网、线粒体),引起细胞内钙离子浓度升高,从而介导凋亡信号通路。④以接种人黑色素瘤细胞A375的BALB/C裸鼠为对象,采用场强20kV/cm、脉宽300ns的高场强纳秒脉冲电场进行处理,通过抑瘤效应观察、透射电子显微镜观察、免疫荧光染色检测Caspase-3蛋白表达、RT-PCR检测Caspase-3 mRNA表达、TUNEL原位末端标记DNA降解等手段,证实了纳秒脉冲电场能有效诱导在体肿瘤凋亡并有效抑制其生长,其机制是通过激活肿瘤细胞Caspase-3蛋白酶而诱导肿瘤细胞调亡,从而使肿瘤组织缩小甚至消失。综上所述,诱导肿瘤细胞凋亡是治疗肿瘤的最新研究热点和可能突破口,而纳秒脉冲电场能有效诱导肿瘤细胞凋亡并明显抑制在体肿瘤组织的生长,在局部肿瘤治疗领域显示出良好的应用前景。更多还原

【Abstract】 Cancer is the main cause of people death. Traditional therapies are not satisfactory due to the limits of appropriateness, contraindication and side effects, etc. In fact, traditional therapies aim at killing caner cells or inhibiting tumor growth, patients can not heal in this way anyway. On the other hand, apoptosis induction could be another way to treat tumor because this way is very effective to make tumor shrink or disappear without any side effects. So, it is very important for tumor treatment to research method and mechanism of apoptosis induction.Based on the traditional Electrical Therapy （ET）, this paper analyzed the common basic bioelectrical mechanism of all kinds of Pulsed Electric Fields （PEF） and then proposed the detailed method and mechanism of Nanosecond Pulsed Electric Fields （nsPEF） for apoptosis induction. The main achievements in this paper are as follows:①Three cell models, Multilayer Dielectric Model （MDM）, Equivalent Circuit Model （ECM） and electric field - electric circuit compound model, were proposed firstly. Calculation methods for transmembrane potential of cell inner and outer membrane of these three models were performed while exposing cancer cells to Electric Field （EF）. Based on the calculation results, Window Effects （WE） of duration - intensity - transmembrane potential, duration - intensity - biomedical effects and conductivity - dielectric constant - transmembrane potential were brought forward, and this supported theory basics for nsPEF to induce cancer cells apoptosis.②A nsPEF generator sample was developed by technical combination of traditional Pulsed Power Technology （PPT）, high power switcher technology and programmable voltage adjusting technology. This sample worked steadily and the parameters of its exponential decay output pulse, peak （0²5kV）, duration （100ns¹μs） and repeat rate （1¹00Hz）, could be adjusted accurately. This supported hardware basics for nsPEF to induce cancer cells apoptosis.③Medical cell experiment to investigate the apoptosis and changes of the intracellular calcium concentration ([Ca²⁺]_i ) induced by nsPEF on human ovarian carcinoma cell line SKOV3 in vitro was carried on. SKOV3 cells were exposed to nsPEF （intensity of 10kV/cm, duration of 100ns, 1 Hz in 5 minutes）. The results showed that early apoptotic rate of experimental group was significantly higher than that of the control group （P<0.01）. The typical morphological features of apoptotic cells were observed by SEM and TEM. It was proved that nsPEF can mainly induced early apoptosis. The [Ca²⁺]_i was markedly increased by treatment with nsPEF （P<0.01）, while the increase showed no relationship to the extracellular calcium concentration （P>0.05）. One of the mechanisms of the apoptosis is that nsPEF can activate intracellular calcium and result in increase of [Ca²⁺]_i so as to induce apoptosis of SKOV3 cells.④Medical animal experiment to research the apoptosis induction effects of nsPEF in vivo was performed. Tumor models in 20 female BALB/C nude mice were established by inoculating with human melanoma cells A375. These mice were randomly divided into treated group （exposed to exponential decay nsPEF with intensity of 20kV/cm, duration of 300ns and 1Hz in 5 minutes） and control group equally. 20 days later, compared with that of control group, tumor growth of treated group was effectively （P<0.01） inhibited, typical morphological apoptosis characteristics in ultrastructure were observed by TEM, expression of Caspase-3 and Caspase-3 mRNA were obviously increased using immunofluorescence and RT-PCR, respectively. These experiment results contributed evidence of tumor growth inhibition by nsPEF exposure in vivo. The mechanism was apoptosis induction effects of nsPEF on cancer cells by activating Caspase-3. This study presented the first in vivo evidence for apoptosis induction effects of nsPEF, and this supported possible tumor therapy utilizing nsPEF.In summary, apoptosis induction is the research focus to treat cancer with great possibility. At the same time, nsPEF is effective to induce cancer cells apoptosis and inhibit tumor growth in vivo, which shows bright future for nsPEF to treat local tumor.更多还原