【作者】 卢翔云；

【导师】 邵吉民；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 苯并(a)芘-7，8-二氢二醇-9，10-环氧化物(BPDE)是多环芳烃类(PAHs)环境化学污染物苯并(a)芘(BaP)在体内的代谢活化产物，并被认为是BaP的终致癌物。BPDE致突变、致癌并不是一个细胞被动接受的过程。BPDE引起大块加成性DNA损伤会激活细胞内核苷酸切除修复、细胞周期阻滞等保护性机制，也会激活低保真度的跨损伤复制机制引发DNA突变，以及激活一些可能有促癌作用的应激信号通路等。因此，要阐明BPDE的致突变、致癌机制，需全面了解细胞对BPDE的应答反应，即基因和毒物的相互作用。我们分别采用Affymetrix HG-U133 Set(～33000个基因)和HG-U133 Plus 2．0(～39000个基因)全基因组芯片来筛选FL人羊膜上皮细胞对不同剂量BPDE(0．005，0．05和0．5μM)处理后同一时间(4 h)的应答基因和同一剂量BPDE(0．05μM)处理后不同时期(1，10和22 h)的应答基因，以较为全面地研究正常人上皮细胞对BPDE的应答反应。基于TaqMan低密度芯片技术对基因芯片结果进行中等通量的定量RT-PCR验证获得了一大批可信的应答基因。剂量效应研究揭示了基因表达(H1F0，AURKA，CCNB1，CENPA，CDC20，KIF14，KIF2C，PKMYT1和EGFR，IGF1R，PRKCA，ITPR1，ATF3，HEXIM1，MYC,SAT1，GDF15，PEA15等)和细胞周期以及细胞毒性表型的紧密关联。转录调控分析结合实验验证发现部分基因的表达改变和BPDE激活一些应激反应相关的转录因子(AP-1，ATF3，NF-κB，p53，Elk-1，CREB和ATF6等)有关，有助于揭示细胞对BPDE应答从应激信号通路，相关转录因子到靶基因的整个过程。个别基因的改变，如CCNE1和CCNE2的上调等可能和BPDE的致癌机制有关。时间效应研究中对细胞周期，细胞生长和凋亡相关基因(H1F0，AURKA，CCCNB1，CENPA，KIF14，NEDD9，SGOL2，RCC1和DLSP1，EIF5A，BIRC4，CTGF，ATF1，JUN，PTEN，CYR61，TOB1，MUC1，FOS，MIRN21等)的分析，揭示了细胞对低浓度BPDE的反应模式，即细胞从启动应答，到整合效应，再到恢复的应答过程。相关基因的改变，如CYR61，MUCl，FOS和MIRN21等基因的上调可能和BPDE的致癌机制有关。对基因芯片结果进行高通量分析发现细胞对BPDE的应答基因涉及广泛的功能包括细胞周期调节，转录调节，RNA剪接、蛋白质代谢、泛素循环、脂类代谢、细胞骨架、细胞内运输、细胞生长、凋亡、信号转导、DNA修复和DNA损伤反应等；以及广泛的信号转导通路如MAPK、黏着斑、细胞周期、Wnt通路和TGFbeta通路等，有助于从全局了解细胞对BPDE的应答机制。对于重要的应答基因和通路与BPDE致突变和致癌机制的关系有待具体的实验研究。更多还原

【Abstract】 (±)-anti-benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is one of the metabolicproducts of benzo(a)pyrene (BaP),which belongs to the polycyclic aromatichydrocarbons (PAHs) environmental chemical pollutants.BPDE is considered as theultimate carcinogen of BaP.BPDE causing mutagenesis and carcinogenesis is not apassively accepted process by the cell.BPDE induces bulky-adduct DNA damage,which would activate intracellular nucleotide excision repair (NER) and cell cyclearrest,etc.protective mechanisms,also trigger the low-fidelity translesion replicationmechanism that leads to DNA mutations,and stimulate some tumor-promotive stresssignaling pathways,etc.Hence,a comprehensive understand of the cellular responseto BPDE,i.e.,the interaction between genes and toxicants,is essential for uncoveringthe mechanisms underlying BPDE-induced mutagenesis and carcinogenesis.We utilized Affymetrix HG-U133 Set (～33000 genes) and HG-U133 Plus 2.0(～39000 genes) whole-genome microarrays,respectively,to obtain responsive genestriggered by various doses (0.005,0.05 and 0.5μM) of BPDE at a same time point (4h) after treatment and by a single dose (0.05μM) of this carcinogen at various timepoints (1,10 and 22 h) after treatment in FL human amnion epithelial cells.Thereby,to rigorously investigate the cellular response to BPDE in normal human cells.Amedium-throughput quantitative RT-PCR validation of the microarray results based on TaqMan Low Density Array has revealed a flock of confident responsive genes.The dose-dependent effect study revealed that gene expression (H1FO,A URKA,CCNB1,CENPA,CDC20,KIF14,KIF2C,PKMYT1 and EGFR,IGF1R,PRKCA,ITPR1,ATF3,HEXIM1,MYC,SAT1,GDF15,PEA15,etc.) and the cell cycle andcytotoxictiy phenotypes were tightly linked.Transcriptional regulatory analysiscombined with experimental validation observed that a part of gene expressionchanges was due to the activation of some stress response-related transcription factors(AP-1,ATF3,NF-κB,p53,Elk-1,CREB and ATF6 etc.) by BPDE treatment.Thushelps to disclose the whole process of cellular response to BPDE from stress signalingpathways,related transcription factors to target genes.The change of a few genes,e.g.,the up-regulation of CCNE1 and CCNE2 might be associated with the mechanism ofBPDE-induced carcinogenesis.Through analysis of the cell cycle,cell growth and apoptosis-related genes (H1FO,A URKA,CCNB1,CENPA,KIF14,NEDD9,SGOL2,RCC1 and DUSP1,EIF5A,BIRC4,CTGF,ATF1,JUN,PTEN,CYR61,TOB1,MUC1,FOS,MIRN21,etc.),the time-courseeffect study revealed a model of celluar response to low-dose BPDE,i.e.,from start ofresponse,to integration and effect-generation,then to recovery.The alteration of somegenes,e.g.,up-regulation of CYR61,MUC1,FOS and MIRN21,etc.,might be relatedwith the mechanism of BPDE-induced carcinogenesis.High throughput analysis of the microarray results showed that the responsivegenes to BPDE were involved in multiple functions including cell cyle regulation,transcription regulation,RNA splicing,protein metabolism,ubiqutin cycle,lipidmetabolism,cytoskeleton,intracellular transport,cell growth,apoptosis,signaltransduction,DNA repair and DNA damage response etc.;and extensive signalingpathways including MAPK,focal adhesion,cell cycle,Wnt signaling pathway andTGFbeta.pathway etc.These observations contribute to a prospective view of thecellular response to BPDE.The relationships between some important responsive genes and pathways and the mechanism of BPDE-induced mutagenesis and carcinogenesisawait concrete experimental investigation in future.更多还原