布雷菲德菌素A诱导人肝癌细胞凋亡的体外研究  

Study on apoptosis of human hepatoma cells induced by brefeldin A

作　　者：高文婷 李明虹 李丹 刘艳飞[1] 李星道 刘希冲 白剑英[1] GAO Wen-ting;LI Ming-hong;LI Dan;LIU Yan-fei;LI Xing-dao;LIU Xi-chong;BAI Jian-ying(Environmental Health,School of Public Health,Shanxi Medical University,Taiyuan,Shanxi 030001,China)

机构地区：[1]山西医科大学公共卫生学院,山西太原030001

出　　处：《毒理学杂志》2023年第5期398-402,408,共6页Journal of Toxicology

基　　金：国家自然科学基金(30972445);山西省自然科学基金(202203021211231)。

摘　　要：目的探讨布雷菲德菌素A(Brefeldin A,BFA)对人肝癌细胞HepG2细胞凋亡的影响。方法用完全培养基(阴性对照组)、0.1%DMSO、0.25 mg/L BFA、1 mg/L衣霉素(Tunicamycin,TM)和0.25 mg/L BFA+1 mg/L TM处理HepG2细胞24 h,MTT法测定细胞活性,流式细胞术检测细胞各组凋亡率,吖啶橙检测试剂盒(AO荧光染色试剂)检测细胞凋亡小体,Western blot法检测细胞内凋亡相关蛋白的表达水平,qRT-PCR法检测细胞内凋亡相关基因的表达。结果形态学观察发现,与阴性对照组相比,0.25 mg/L BFA组细胞明显变得稀疏。细胞活性实验发现0.25 mg/L BFA组,0.25 mg/L BFA与1 mg/L TM联合处理组细胞存活率均明显降低(F=59.766,P<0.05)。流式细胞仪分析发现,0.25 mg/L BFA组,0.25 mg/L BFA与1 mg/L TM联合处理组细胞早期凋亡率明显升高(F=20.738,P<0.05)。AO凋亡小体检测发现,0.25 mg/L BFA组和0.25 mg/L BFA+1 mg/L TM联合组人肝癌细胞HepG2细胞内出现核固缩现象。从蛋白水平来看,与对照组相比,0.25 mg/L BFA组和0.25 mg/L BFA+1 mg/L TM联合组硬酯酰辅酶A去饱和酶1(stearoyl-CoA desaturase 1,SCD1)蛋白表达明显减少,甚至无法检出,聚腺苷二磷酸-核糖聚合酶-1(poly-ADP-ribose polymerase-1,PARP-1)、半胱氨酸蛋白酶12(cysteine aspartic acid specific protease-12,Caspase-12)蛋白表达升高,天冬氨酸特异性半胱氨酸蛋白酶3(cysteine aspartic acid specific protease-3,Caspase-3)活性形式cleaved Caspase-3被检出。基因检测结果显示,与对照组相比,0.25 mg/L BFA+1 mg/L TM联合组SCD1 mRNA表达水平升高(F=19.229,P<0.05);0.25 mg/L BFA组和0.25 mg/L BFA+1 mg/L TM联合组PARP-1、Caspase-12和Caspase-3 mRNA表达水平均明显升高(F=627.475,F=68.640,F=238.742,P<0.05)。结论0.25 mg/L BFA可抑制SCD1蛋白表达,且促进人肝癌细胞凋亡,1 mg/L TM可增强其凋亡诱导作用。Objective To investigate the effect of brefeldin A(BFA)on apoptosis of HepG2 cells.Methods HepG2 cells were treated with 0.1%DMSO,0.25 mg/L BFA,1 mg/L TM and 0.25 mg/L BFA+1 mg/L TM for 24 h.Cell viability was determined by MTT assay,and the effect on apoptosis rate was detected by flow cytometry,AO fluorescent staining reagent was used to detect apoptotic bodies,western blot was used to detect the expression levels of apoptosis-related proteins,and qRT-PCR was used to detect the expression levels of apoptosis-related genes.Results Compared with the control group,cells in BFA became sparse.Cell viability test showed that the survival rate of BFA and TM+BFA combined group were significantly reduced.The early apoptosis rate of BFA and TM+BFA combined group were significantly increased.AO apoptotic bodies detection showed that there were pyknosis of the nucleus in HepG2 cells of BFA and BFA+TM combined group.In terms of protein levels,compared with the control group,protein expressions of SCD1 were significantly reduced,and even could not be detected in HepG2 cells of BFA and BFA+TM combined group,protein expressions of PARP-1,Caspase-12,Caspase-3 and cleaved Caspase-3 were increased in BFA and BFA+TM combined group,compared with the control group,the mRNA expression levels of SCD1 in BFA+TM combined group were increased,the mRNA expression levels of PARP-1,Caspase-12 and Caspase-3 in BFA and BFA+TM combined group were significantly increased.Conclusion BFA can inhibit the expression of SCD1 protein and promote the apoptosis of HepG2 cells,and TM can enhance the apoptosis induction.

关 键 词：布雷菲德菌素A 肝癌细胞 硬脂酰辅酶A去饱和酶1 细胞凋亡 半胱氨酸蛋白酶12 

分 类 号：R73-36[医药卫生—肿瘤] R114[医药卫生—临床医学] R99