Yin Shengyong, Chen Xinhua, Xie Haiyang, Zhou Lin, Guo Danjing, Xu Yuning, Wu Liming, Zheng Shusen
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou, China; Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China.
Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China.
Exp Cell Res. 2016 Aug 15;346(2):233-40. doi: 10.1016/j.yexcr.2016.06.018. Epub 2016 Jun 30.
Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.
先前的研究表明，纳秒级脉冲电场（nsPEF）可消融包括肝细胞癌（HCC）在内的实体瘤，但其对细胞膜的作用尚未完全明确。我们推测，nsPEF通过直接机械力破坏细胞膜外微区，进而导致质膜通透性增加，促进小分子摄取。对三种肝癌细胞每分钟施加一个脉冲，该间隔长于纳米孔重新封闭的时间。用阳离子铁蛋白标记电负性微区，用碘化丙啶（PI）检测膜通透性，用能量色散X射线光谱（EDS）检测细胞表面负电荷，用顺铂检测细胞内细胞毒性。我们证明，nsPEF产生的机械力破坏了铁蛋白标记的微区和细胞表面负电荷。PI的细胞摄取和DNA靶向顺铂的细胞毒性呈剂量依赖性增加。顺铂与nsPEF刺激联合使用时，细胞内细胞毒性达到最大。我们得出结论，nsPEF直接破坏细胞膜外微区，增加PI和顺铂的膜通透性。微区破坏和膜渗透变化是由细胞表面负电荷变化产生的机械力引起的。
