二羟环氧苯并芘短期暴露对人支气管上皮细胞基因表达谱的影响  

Effects on gene expression profile in human bronchial epithelial cells exposed to benzo[a]pyrene diol epoxide

作　　者：王慧[1] 吕懿[1] 李玲 李朝菲 郭璨璨 田凤[1] 穆箭兵 郑金平[1,3] WANG Hui;LYU Yi;LI Ling;LI Zhaofei;GUO Cancan;TIAN Fengjie;MU Jianbing;ZHENG Jinping(Department of Health Toxicology,School of Public Health,Shanxi Medical University,Taiyuan,Shanxi 030001,China;National Institutes of Health(NIH),Bethesda,Maryland 20892,USA;Department of Public Health and Preventive Medicine,Changzhi Medical College,Changzhi,Shanxi 046000,China)

机构地区：[1]山西医科大学公共卫生学院卫生毒理学教研室,山西太原030001 [2]美国国立卫生研究院,马里兰州贝塞斯达20892 [3]长治医学院公共卫生与预防医学系,山西长治046000

出　　处：《环境与职业医学》2021年第1期51-57,63,共8页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(30872137);山西省重点研发计划国际科技合作项目(201703D421021);山西省青年科技研究基金(201901D211327)。

摘　　要：[背景]苯并[a]芘(BaP)可引起暴露人群呼吸系统损伤。既往研究表明,BaP及其活性代谢物染毒细胞24h,即可引发细胞能量代谢异常、氧化损伤,导致细胞生存率降低。[目的]观察BaP活性代谢物二羟环氧苯并芘(BPDE)短期暴露导致的人支气管上皮细胞(16HBE)基因表达谱的变化,为探讨BPDE急性毒性机制提供依据。[方法]选取人支气管上皮细胞(16HBE),分别用终浓度0.5、1.0、2.0、4.0μmol·L^(-1)的BPDE染毒,同时设溶剂对照组(加入等体积二甲基亚砜)和空白对照组(加入正常培养基),处理24h后,CCK8法检测细胞存活率。根据细胞存活率,挑选一个剂量染毒组和溶剂对照组利用BGISECQ平台进行转录组测序(RNA-Seq)。使用R语言中的DEseq2包检测不同样本的差异表达基因,并用phyper函数对差异基因进行基因本体论(GO)功能注释和京都基因与基因组百科全书(KEGG)通路分析。[结果]随BPDE染毒剂量的增加,16HBE细胞存活率呈下降趋势(Z=-7.79,p趋势<0.01);0.5、1.0、2.0、4.0μmol·L^(-1) BPDE染毒组细胞存活率分别为94.56%±1.74%、84.80%±3.19%、80.08%±1.72%、62.72%±1.95%,均明显低于溶剂对照组(98.61%±0.61%)和空白对照组(100.00%±0.00%)(P<0.05)。选取2.0μmol·L^(-1)BPDE染毒组和溶剂对照组细胞进行测序,结果显示,以基因变化倍数的对数绝对值|log2FC|>1,P<0.05为筛选标准,暴露组共检测出191个2倍及以上差异表达的基因;其中上调的基因87个,下调的基因104个。基因共表达网络分析发现,趋化因子CXCL6、CXCL1、CXCL3、CXCL8和集落刺激因子CSF2具有较强的关联性。GO分析显示,差异表达基因主要参与细胞增殖的生物学过程,并通过KEGG通路分析发现,这些基因主要富集于白介素(IL)-17信号通路和炎症趋化因子等通路。进一步分析差异表达的mRNA,发现有670个mRNA表达上调,878个mRNA表达下调;差异表达mRNA主要富集于凋亡执行期负调控、细胞外信号转导负调[Background]Benzo[a]pyrene(B[a]P)can cause respiratory damage in people exposed to it.Previous studies indicate that a 24-hour exposure to B[a]P and its active metabolites could cause abnormal cell ene rgy metabolism and oxidative damage,leading to reduced cell survival.[Objective]This experiment observes the changes of gene expression profile of human bronchial epithelial cells(16 HBE)caused by short-term exposure to benzo[a]pyrene diol epoxide(BPDE),an active metabolite of B[a]P,and to provide evidence for the potential mechanism of acute toxicity of BPDE.[Methods]This experiment included four BPDE exposure groups(0.5,1.0,2.0,and 4.0μmol·L^(-1),respectively),a solvent control group(dimethyl sulfoxide),and a blank control group(normal saline).CCK8 method was used to measure cell survival rate after 24 h treatment.Acco rding to the cell survival rate,an exposure group and a solvent control group were selected for transcriptional sequencing(RNA-Seq)using the BGISEQ platform.The DEseq2 package in R software was used to detect the differentially expressed genes(DEGs)in different samples.The phyper function in R software was used to perform Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of the selected DEGs.[Results]The survival rates of 16 HBE cells decreased with the increase of BPDE concentration(Z=-7.79,Ptrend<0.01).The cell survival rates of the 0.5,1.0,2.0,and 4.μmol·L^(-1) BPDE groups(94.56%11.74%,84.80%±3.19%,80.08%±1.72%,and 62.72%11.95%,respectively)were significantly reduced compared with the solvent control group(98.61%±0.61%)and the blank control group(100.00%±0.00%)(P<0.05).The cells in the 2.0 pmol·L^(-1) BPDE group and the solvent control group were selected for sequencing,and 191 DEGs satisfying both|log2 FC|>1 and P<0.05 were detected in the exposure group,including 87 genes up-regulated and 104 genes down-regulated.The results of gene co-expression network found that chemokines CXCL6,CXCL1,CXCL3,and CXCL8 had strong corre

关 键 词：二羟环氧苯并芘 苯并[A]芘 短期暴露 mRNA测序 生物信息学分析 

分 类 号：R114[医药卫生—卫生毒理学]