微秒脉冲电场促进肝癌细胞凋亡及其机制  

Promotion of apoptosis and mechanisms in hepatocellular carcinoma cells by microsecond pulsed electric field

作　　者：郑伟 陈新华 胡深 黄琦 Zheng Wei;Chen Xinhua;Hu Shen;Huang Qi(Dept of Oncology,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;Zhejiang Provincial Key Laboratory of Pulsed Electric Field Technology for Medical Transformation,Hangzhou 311121;Dept of Obstetrics,The Second Affiliated Hospital of Zhejiang University School of Medicine,Hangzhou 310052)

机构地区：[1]安徽医科大学第二附属医院肿瘤科,合肥230601 [2]浙江省脉冲电场技术医学转化重点实验室,杭州311121 [3]浙江大学医学院附属第二医院产科,杭州310052

出　　处：《安徽医科大学学报》2024年第8期1370-1376,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:82003037);安徽省转化医学研究院科研基金(编号:2022zhyx-C79);浙江省科技健康计划(编号:2021KY719)。

摘　　要：目的 初步探究微秒脉冲电场引起肝癌细胞HepG2凋亡的现象及其机制。方法 利用CCK-8检测不同参数的微秒脉冲电场对人肝癌细胞HepG2和人正常肝细胞L-O2增殖的影响,通过流式细胞术观察不同电场强度下微秒脉冲消融对HepG2细胞凋亡的影响;利用透射电镜观察消融后的HepG2细胞形态改变;通过转录组测序和生物信息学分析筛选消融组和对照组细胞间的差异表达基因;采用实时荧光定量PCR(qPCR)实验和蛋白质印迹实验验证HepG2细胞经脉冲消融后差异表达基因的RNA和蛋白表达改变。结果 CCK-8实验显示,随着电场强度的增加,微秒脉冲电场消融对肝癌细胞HepG2和人肝细胞L-O2的增殖和抑制能力也逐渐增加。但微秒脉冲电场对二者之间的消融能力没有显著性差异。在电压增加至1 600 V/cm时,微秒脉冲电场能够诱导HepG2细胞凋亡(P<0.000 1),凋亡细胞比率近80%。透射电镜观察显示,微秒脉冲电场消融后,HepG2细胞膜发生破碎,其中线粒体形态不规则,出现了典型的凋亡小体。转录组学分析揭示参与微秒脉冲消融的主要基因有丝氨酸苏氨酸蛋白激酶1(TAOK1),卷曲蛋白家族受体3(FZD3),钙蛋白酶10(CAPN10),基质金属蛋白酶9(MMP-9)以及转化生长因子β-1诱导的转录本(TGFB1)等。qPCR实验证实在1 600 V/cm微秒脉冲消融后,HepG2细胞内TAOK1(P<0.05)、FZD3(P<0.01)的RNA表达水平上调,CAPN10(P<0.001)、MMP-9(P<0.01)和TGFB1(P<0.05)的RNA表达水平降低。蛋白质印迹实验进一步证实TAOK1和FZD3在微秒脉冲处理后表达水平出现上升,而CAPN10、MMP-9和TGFB1的表达水平出现下调。结论 微秒脉冲电场消融可以有效促进肝癌HepG2细胞的凋亡,CAPN10、FZD3等基因可能参与微秒脉冲消融促进肝癌HepG2细胞凋亡的调控。Objective To preliminarily explore the phenomenon and mechanism of apoptosis induced by microsecond pulsed electric field in hepatocellular carcinoma cells HepG2.Methods CCK-8 was used to test the effects of microsecond pulsed electric fields with different parameters on the proliferation of human hepatocellular carcinoma cells HepG2 and human normal hepatocytes L-O2.The effects of microsecond pulsed electric field ablation on HepG2 cell apoptosis under different electric field intensities were observed by flow cytometry;morphological changes of ablated HepG2 cells were observed by transmission electron microscopy;differentially expressed genes between ablated and control cells were screened by transcriptome sequencing and bioinformatics analysis,and the RNA and protein expression of differentially expressed genes in HepG2 cells after pulse ablation was verified by quantitative real-time PCR(qPCR)and Western blot.Results CCK-8 experiments showed the proliferative and inhibitory abilities of microsecond pulsed electric field ablation on hepatocellular carcinoma cells HepG2 and human hepatocytes L-O2 increased gradually with the increase of electric field intensity.However,there was no significant difference in the ablation ability of microsecond pulsed electric field between HepG2 cell and L-O2 cell.When the voltage was increased to 1600 V/cm,the microsecond pulsed electric field significantly induced apoptosis in HepG2 cells compared to control group,with an apoptosis cells ratio of nearly 80%(P<0.0001).Using transmission electron microscopy,it was found that the HepG2 cell membrane was fragmented,the mitochondria were irregular in morphology and typical apoptotic vesicles appeared after microsecond pulsed electric field ablation.Transcriptomics analysis showed the major genes revealed to be involved in microsecond pulse ablation were thousand and one amino acid protein kinase 1(TAOK1),frizzled protein family receptor 3(FZD3),calpain 10(CAPN10),matrix metalloproteinase 9(MMP-9),and transforming growth factorβ-1-

关 键 词：肝癌 微秒脉冲消融 凋亡 

分 类 号：R735.7[医药卫生—肿瘤]