自制的纳秒脉冲电场（nsPEF）肿瘤消融设备，已证实能安全有效清除实体瘤。除直接致细胞膜穿孔死亡外，nsPEF通过库伦力作用于细胞内带电荷的分子，将Calreticulin和PD-L1等分子拖曳到细胞外膜，改变肿瘤细胞与免疫细胞的相互应答。我们假定nsPEF消融肝癌激活的肿瘤免疫会被PD-L1耗竭。本项目拟利用nsPEF处理不同来源的肝癌细胞系，结合蛋白质谱分析等技术处理前后细胞膜PD-L1等关键分子的含量变化，进而分析PD-L1空间位置变化引起的免疫效应改变。动物体内研究nsPEF消融引起的PD-L1分子变化对肿瘤细胞周围及血液循环中CD8+ T细胞的细胞毒性功能和耗竭的影响。进而研究nsPEF消融联合抗PD-L1免疫治疗对荷瘤鼠肿瘤特异性CD8T+细胞亚群数量和功能的影响，及综合治疗对延长小鼠长期生存的意义。为原创的nsPEF肿瘤消融设备应用于临床肝癌综合治疗提供新思路。

A home-made experimental equipment to ablate solid cancer with nanosecond pulsed electric field，had been validated to eliminate solid tumors safely and effectively. NsPEF acts on charged molecules inside of plasma membrane through Coulomb force, and then draws intracellular molecules such as Calreticulin and PD-L1 and delivers them onto extracellular leaflet of plasma membrane. As a result of spatio-temporal change of Calreticulin and PD-L1, the mutual response of immune cells to tumor cells changed. We hypothesized that nsPEF activities tumor immune response to ablated hepatocellular Carcinoma cells but it would be exhausted by PD-L1. In this study, nsPEF applies to treat Hepatocellular carcinoma cell lines from different sources. Contents change of key molecules such as PD-L1 before and after nsPEF investigates with technologies including protein mass spectrometry. Further study focuses on changes of immune responses induced by spatio-temporal change of PD-L1. In vivo study investigates effects on cytotoxicity and exhaustion of CD 8+ T cells in microenviroment of ablated tumor tissue and of blood circulation in tumor burden mice by nsPEF induced PD-L1 change. Further studies concentrated on influences to number and function of tumor specific CD 8+ T cells population of comprehensive therapy treated HCC that combined nsPEF ablation with anti-PD-L1 therapy, and on the significance of comprehensive therapy for prolonging the survival of mice. This research provides new ideas for the translation of nsPEF to clinical liver cancer therapy.