砷及其主要代谢产物对A549细胞凋亡及促凋亡基因Bad和Bik表达的影响  

Effects of arsenic and its main metabolites on A549 cell apoptosis and the expression of pro-apoptotic genes Bad and Bik

作　　者：周倩 尹锦瑶 谭婧文 李舒婷 蒋成兰 何越峰 Zhou Qian;Yin Jinyao;Tan Jingwen;Li Shuting;Jiang Chenglan;He Yuefeng(School of Public Health,Kunming Medical University,Kunming 650500,China)

机构地区：[1]昆明医科大学公共卫生学院,昆明650500

出　　处：《中华劳动卫生职业病杂志》2022年第9期661-667,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金(81860572);2020年云南省科技厅昆明医科大学应用基础研究联合专项青年博士项目(202001AY070001-135)。

摘　　要：目的探讨砷及其主要代谢产物对人肺腺癌细胞系A549细胞凋亡及促凋亡基因Bad和Bik表达的影响。方法于2020年10月,复苏培养A549细胞,利用细胞计数试剂CCK-8检测细胞活力,确定亚砷酸钠(NaAsO_(2))染毒A549细胞的浓度和时间。研究分为NaAsO_(2)染毒组和代谢产物染毒组:NaAsO_(2)染毒组染毒剂量为0(对照组)、20、40、60μmol/L;代谢产物染毒组包括60μmol/L一甲基胂酸(MMA)染毒组、60μmol/L二甲基胂酸(DMA)染毒组。采用Hoechst33342/碘化丙啶双染法(Ho/PI)观察细胞凋亡情况,采用实时荧光定量聚合酶链式反应法(qRT-PCR)检测染毒后细胞Bad和Bik mRNA表达水平,采用蛋白免疫印迹法(Western blotting)检测Bad蛋白、磷酸化Bad(P-Bad-S112)蛋白、Bik蛋白、裂解Bik蛋白及下游蛋白多聚腺苷二磷酸核糖聚合酶(PARP1)和细胞色素C(Cyt-C)的相对表达情况,采用分光光度法检测半胱氨酸天冬氨酸特异性蛋白酶(Caspase)3、6、8、9的活性变化。结果与对照组比较,20、40、60μmol/L NaAsO_(2)染毒组凋亡细胞占比明显增加,差异均有统计学意义(P<0.01)。与对照组比较,2.0、4.0、6.0μmol/LNaASO_(2)染毒组Bad mRNA表达水平、Bik mRNA表达水平升高,Bad蛋白、磷酸化Bad蛋白、Bik蛋白、裂解Bik蛋白、PARP1蛋白、Cyt-C蛋白相对表达量均升高,差异均有统计学意义(P<0.05),Caspase 3、6、8、9活性明显上调,差异均有统计学意义(P<0.05)。与对照组比较,DMA染毒组Bad mRNA表达水平为1.439±0.173,差异有统计学意义(P=0.024),Bik mRNA表达水平差异无统计学意义(P=0.788);MMA染毒组Bad和Bik mRNA表达水平差异无统计学意义(P=0.085、0.063)。与对照组比较,MMA染毒组Bad、Bik、PARP1、Cyt-C蛋白相对表达量(0.696±0.023、0.707±0.014、0.907±0.031、1.032±0.016)差异无统计学意义(P=0.469、0.669、0.859、0.771);DMA染毒组Bad、Bik、PARP1、Cyt-C蛋白相对表达量(0.698±0.030、0.705±0.022、0.908±0.015、1.029±0.010)差�Objective To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik.Methods In October 2020,A549 cells were recovered and cultured,and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549.The study was divided into NaAsO_(2) exposure groups and metobol:le expoure groups:the metabolite comparison groups were subdivided into the control group,the monomethylarsinic acid exposure group(60μmol/L),and the dimethylarsinic acid exposure group(60μmol/L);sodium arsenite dose groups were subdivided into 4 groups:control group(0),20,40,60μmol/L sodium arsenite NaAsO_(2).Hoechst 33342/propidium iodide double staining(Ho/PI)was used to observe cell apoptosis and real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of Bad and Bik mRNA in cells after exposure.Western blotting was used to detect the protein expressions of Bad,P-Bad-S112,Bik,cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C(Cyt-C),using spectrophotometry to detect the activity changes of caspase 3,6,8,9.Results Compared with the control group,the proportion of apoptotic cells in the 20,40,and 60μmol/L NaAsO_(2) dose groups increased significantly(P<0.01),and the expression levels of Bad,Bik mRNA,the protein expression levels of Bad,P-Bad-S112,Bik,cleaved Bik,PARP1,Cyt-C were increased(all P<0.05),and the activities of Caspase 3,6,8,and 9 were significantly increased with significantly differences(P<0.05).Compared with the control group,the expression level of Bad mRNA in the DMA exposure group(1.439±0.173)was increased with a significant difference(P=0.024),but there was no significant difference in the expression level of Bik mRNA(P=0.788).There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups(P=0.085,0.063).Compared with the

关 键 词：砷 代谢产物 细胞凋亡 Bad基因 Bik基因 亚砷酸钠 一甲基胂酸 二甲基胂酸 

分 类 号：R114[医药卫生—卫生毒理学]