基于微秒脉冲调制高频纳秒脉冲电场诱导凋亡并激发不可逆电穿孔效应治疗肿瘤的基础研究

In order to solve the problems involved in tumor treatment using pulsed electric fields (PEF), this project presented a novel tumor treatment method using high-frequency nanosecond pulsed electric fields (nsPEF) modulated by microsecond pulse based on both apoptosis and irreversible electroporation, also aiming at direct killing effects of microsecond pulsed electric fields (μsPEF) and indirect modulating effects of nsPEF. Firstly, a equivalent circuit model of spherical cell with the consideration of cellular structure dispersion was established and used to calculate transmembrane potentials of plasma membrane and intracellular membrane to obtain the minimum pulse parameters. A dielectric model of tumor tissue was also established and used to assess the thermal effects of this new method to get the maximum pulse parameters. As a result, a range of appropriate pulse parameters for this method was achieved. After that, a transport lattice model of cancer cell with nucleus and mitochondria, introducing a voltage controlled current source into its equivalent circuit, was proposed to thoroughly investigate the regulations of synergistic and cumulative electroporation of cancer cells exposed to the new pulse in this project. Finally, cell experiment in vitro and animal experiment in vivo were carried out to explore the lethal machanisms of cancer cells possibly associated with both apoptosis and irreversible electroporation. The efficacy and safety of this novel method were also verified by these medical experiments. In conclusion, principle and technology of this new method presented in this project would provide theoretical basis and experimental evidence for development of new medical equipment based on high-frequency nsPEF modulated by microsecond pulse with important academic value and good prospects for clinical application.

为解决现有脉冲电场治疗肿瘤技术存在的问题，并同时发挥微秒脉冲电场（μsPEF）的直接杀伤作用和纳秒脉冲电场（nsPEF）的间接调控作用，本项目提出基于微秒脉冲调制高频nsPEF诱导凋亡并激发不可逆电穿孔效应治疗肿瘤的创新思路。建立考虑频率色散效应的肿瘤细胞等效电路模型和肿瘤组织介电模型，通过细胞内外膜跨膜电位理论计算和热效应评估，确定脉冲窗口参数；建立包含细胞核和线粒体的肿瘤细胞微观传输网格模型，在网格等效电路中引入压控电流源，深入研究微秒脉冲调制高频nsPEF的协同穿孔规律和累积穿孔规律；开展离体细胞实验和荷瘤动物实验，研究微秒脉冲调制高频nsPEF诱导凋亡并激发不可逆电穿孔的全面杀伤机理，并验证其有效性与安全性。本项目旨在揭示微秒脉冲调制高频nsPEF治疗肿瘤技术的原理和方法，为最终成功开发拥有自主知识产权的新型医疗设备提供理论基础和实验依据，具有重要的学术价值和良好的临床应用前景。

为解决现有脉冲电场治疗肿瘤技术存在的问题，并同时发挥微秒脉冲电场的直接杀伤作用和纳秒脉冲电场的间接调控作用，本项目对基于微秒脉冲调制高频纳秒脉冲电场（高频纳秒脉冲串）诱导凋亡并激发不可逆电穿孔效应治疗肿瘤的创新方法进行了深入研究。基于印刷电路板Blumlein传输线、传输线变压器等拓扑结构，结合多个固态开关串并联技术和高频脉冲调制技术，研究了模块化固态高压高频纳秒脉冲（幅值0～10kV、脉宽50～500ns、重复频率1Hz～1MHz）产生技术；建立肝脏肿瘤和皮肤肿瘤的介电模型，采用有限元方法分析了肿瘤组织热效应与脉冲参数的关系，得到了高频纳秒脉冲串作用下肿瘤组织温度与热损伤的时空分布，以及肿瘤组织不发生热损伤的脉冲参数范围，并用荷瘤裸鼠进行了活体动物实验验证；分别建立单个肿瘤细胞的网格传输网络模型和多个肿瘤细胞的笛卡尔传输网格模型，采用动态电穿孔数学模型对肿瘤细胞的动态穿孔过程和分子跨膜传输特性进行了仿真计算，得到了高频纳秒脉冲串作用下肿瘤细胞空间电位分布、跨膜电压变化、孔密度/孔半径分布以及分子浓度空间分布和跨膜通量，发现高重复频率的多个纳秒脉冲会使肿瘤细胞内外膜孔数量/孔半径和分子跨膜传输体现出明显的累积效应；以离体人黑色素瘤细胞、荷瘤裸鼠、新西兰大白兔为对象，开展医学实验研究高频纳秒脉冲串对肿瘤细胞活性、凋亡率/坏死率、迁移能力、细胞克隆能力、细胞周期、抑瘤效果、肌肉收缩的影响，发现高频纳秒脉冲串能诱导凋亡并激发不可逆电穿孔而有效杀伤肿瘤细胞，并能有效抑制在体肿瘤生长，适当范围的脉冲参数能保证治疗过程的安全性。综上，本项目揭示了高频纳秒脉冲串杀伤肿瘤细胞的累积穿孔机理及其安全有效杀伤肿瘤细胞的方法，显示出良好的临床应用前景。