p38MAPK基因沉默对PM2.5染毒肝细胞后癌基因和凋亡基因表达的影响  

Effect of p38MAPK gene silencing on expression of oncogenes and apoptotic genes induced by PM2.5 in hepatocytes

作　　者：王冰玉 秦双建 李闰冰 李柏茹 蔡颖[1,2] 郑凯[1,2] 徐新云 谢红卫[2] Wang Bingyu;Qin Shuangjian;Li Runbing;Li Boru;Cai Ying;Zheng Kai;Xu Xinyun;Xie Hongwei(Institute of Environment and Health,Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,China;School of Public Health,University of South China,Hengyang 421001,China;Xiangya School of Public Health,Central South University,Changsha 410078,China)

机构地区：[1]深圳市疾病预防控制中心环境与健康所,518055 [2]南华大学公共卫生学院,衡阳421001 [3]中南大学湘雅公共卫生学院,长沙410078

出　　处：《中华劳动卫生职业病杂志》2020年第10期721-725,共5页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：深圳市科技研发基础研究项目(JCYJ20170413101713324)。

摘　　要：目的研究p38丝裂原活化蛋白激酶(MAPK)基因沉默对大气细颗粒物(PM2.5)染毒肝细胞(L02细胞)相关基因表达的影响。方法于2019年6至9月,根据GenBank提供的p38MAPK基因序列,设计合成3条干扰序列,退火后连接到PLVX-shRNA2-puro中,将重组慢病毒载体转染L02细胞。用实时荧光定量聚合酶链反应(PCR)和蛋白免疫印迹(Western blotting)法对p38MAPK基因沉默效果进行鉴定。用浓度为50μg/ml的PM2.5水溶物和10μmol/L阳性对照Cr^6+分别染毒L02细胞、p38MAPK基因沉默细胞24 h,同时设立空白对照组。用荧光定量PCR检测癌基因(c-fos、c-myc、k-ras)、抑癌基因(p53)和凋亡基因(Caspase-3、Caspase-8、Caspase-9)的相对表达水平。结果p38MAPK基因沉默细胞中p38MAPK基因和蛋白表达水平较正常L02细胞明显下降(P<0.05),p38MAPK基因沉默细胞株构建成功。与空白对照组比较,PM2.5染毒L02细胞组癌基因c-fos、c-myc、k-ras和凋亡基因Caspase-3、Caspase-8、Caspase-9表达水平升高,抑癌基因p53表达水平下降,差异均有统计学意义(P<0.05);与PM2.5染毒L02细胞组比较,PM2.5染毒p38MAPK基因沉默细胞组癌基因c-fos、c-myc、k-ras和凋亡基因Caspase-3、Caspase-8、Caspase-9表达水平下降,抑癌基因p53表达水平升高,差异均有统计学意义(P<0.05)。结论PM2.5对L02细胞癌基因、抑癌基因和凋亡基因表达有明显影响,而p38MAPK基因沉默可抑制PM2.5对L02细胞的影响。Objective To study the effect of p38 mitogen-activated protein kinase(MAPK)gene silencing on expression of apoptotic genes and oncogenes in hepatocytes treated with PM2.5.Methods From June to September 2019,according to the p38MAPK gene mRNA sequence provided by GenBank,three interfering sequences were designed and synthesized,ligated into PLVX-shRNA2-puro after annealing,and the recombinant lentiviral vector was transfected into L02 hepatocytes.The p38MAPK silencing cells were identified by real-time fluorescent quantitative PCR and western blotting.The normal L02 cells and p38MAPK silencing cells were treated with 50μg/mL PM2.5 water soluble solution,10μmol/L positive control Cr^6+,and a blank control group was set up,the treatment time was 24 h.The mRNA levels of oncogenes(c-fos,c-myc,k-ras),tumor suppressor gene(p53)and apoptotic genes(Caspase-3,Caspase-8,Caspase-9)were detected by real-time PCR.The protein levels of oncogenes and apoptotic genes were detected by Western blotting.Results The expression levels of p38MAPK mRNA and protein in p38MAPK gene silencing cells were significantly lower than those in L02 hepatocytes(P<0.05),and the p38MAPK gene silencing cell line was successfully constructed.Compared with the blank control group,the expression levels of the oncogenes c-fos,c-myc,k-ras and the apoptosis genes Caspase-3,Caspase-8 and Caspase-9 increased,the expression level of tumor suppressor gene p53 decreased in the L02 hepatocyte group treated with PM2.5 water soluble matter,and the differences were statistically significant(P<0.05).Compared with the L02 hepatocytes group treated with PM2.5 water soluble matter,the expression levels of the oncogenes c-fos,c-myc,k-ras and apoptosis genes Caspase-3,Caspase-8 and Caspase-9 decreased,the expression level of tumor suppressor gene p53 increased in the p38MAPK gene silencing cells group treated with PM2.5 water soluble matter,and the differences were statistically significant(P<0.05).Conclusion PM2.5 has effects on the expression of oncogenes,tumor suppre

关 键 词：肝细胞 基因沉默 细颗粒物 雾霾 凋亡基因 癌基因 

分 类 号：R114[医药卫生—卫生毒理学]