高频双极性脉冲诱导SKOV–3细胞生物电效应的实验研究

Experimental Study on Bioeffect of SKOV–3 Cell Induced by Bipolar High Frequency Pulses

不可逆电穿孔技术以其非热、微创的独特优势而广泛应用于肿瘤治疗领域,但在治疗过程中会出现消融不彻底与肌肉收缩这2个问题;高频双极性脉冲作为新式脉冲形式可在肿瘤组织中产生均匀电场来解决肌肉收缩难题,同时与传统脉冲相比能有效缓解肌肉收缩。为此,以SKOV–3细胞为研究对象,采用CCK–8检测细胞活性,采用JC–1荧光探针分子观察线粒体膜电位变化,采用免疫组化技术检测凋亡蛋白表达。研究发现：传统单极性脉冲对肿瘤细胞的杀伤机理主要是对细胞膜的不可逆破坏,继而导致细胞坏死;ns高频双极性脉冲虽然与传统脉冲具有相同的电场强度与高电平时间,但却不能有效杀死肿瘤细胞;而μs高频双极性脉冲不仅能有效诱导细胞坏死,而且通过活性检测发现也可能诱导细胞凋亡;进一步研究发现,μs高频双极性脉冲能作用于线粒体使线粒体膜电位崩溃;同时也检测出BAX、BCL–2、Caspase–3和Caspase–9凋亡蛋白的表达。该研究结果表明μs高频双极性脉冲不仅可诱导细胞坏死,而且可启动线粒体凋亡程序来杀死细胞。

Irreversible electroporation（IRE） has developed a non-thermal and minimally invasive method for tumor treatment. However, the unexhausted tumor ablation and muscle contraction could happen during IRE treatment. Bipolar high frequency pulses, a new modality of pulses can produce a more homogeneous electric field in heterogeneous tissues to solve the unexhausted ablation. This modality also relieves muscle contraction as traditional IRE pulses. However, the mechanism of cell killing by bipolar high frequency pulses is still not clear. We stimulated SKOV-3 cells by bipolar high frequency pulses to study the mechanism of cell killing. The CCK-8 was used to measure the cell viability. The fluorescent molecular probe JC-1 was used to measure the mitochondrial membrane potential. The fluorescent molecular probe JC-1 was used to measure the mitochondrial membrane potential. The immunohistochemical techniques were used to measure the expression of apoptosis proteins. The results showed that traditional IRE pulses could kill cells by permanently disrupting cell membrane. Although nanosecond bipolar high frequency pulses have the same electric field and duration as IRE pulses, the tumor cell could not be killed effectively. However, microsecond bipolar high frequency pulses not only make tumor cells necrosis, but also start the tumor cell apoptosis process. Moreover, microsecond bipolar high frequency pulses exhibit a strong inward collapse of mitochondrial membrane potential. The expression of BCL–2, BAX, Caspase–3, and Caspase–9 apoptosis proteins are also detected. Therefore, the microsecond bipolar high frequency pulses not only made cell necrosis but also disrupted mitochondria and activated the apoptosis process.