Effects of three polycyclic aromatic hydrocarbons:benzo(a)pyrene, pyrene and phenanthrene on embryo development of Sebastiscus marmoratus

多环芳烃(Polycyclicaromatichydrocarbons，PAHs)是海洋中常见的污染物，极易在环境中累积并可通过食物链传递，对人类健康和生态环境具有很大危害。已有研究表明PAHs具有致畸、致癌、致突变、免疫抑制等作用。本研究利用环境水平（0.5、5、50nmol/L）三环的菲(phenanthrene，Phe)和四环的芘(pyrene，Pyr)以及（0.5、5、25nmol/L）五环的苯并(a)芘[benzo(a)pyrene，BaP]分别暴露褐菖鮋胚胎，研究PAHs对鱼类胚胎发育的毒性。 PAHs暴露囊胚期胚胎7天，其中0.5和25nmol/LBaP组死亡率显著高于对照组；...Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants being found in aquatic areas. Exposure to PAHs is linked to teratogenesis, carcinogenesis, mutagenesis, immunosuppression and other numerous health problems. In the present study, Sebastiscus marmoratus embryos exposure to benzo(a)pyrene (BaP, 5-ring), pyrene (Pyr, 4-ring) and phenanthrene (Phe, 3-ring) at environme...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：2172009015353

Pregnane X receptor involves in the effect of aflatoxin B1 on necroptosis in human normal L02 liver cells

目的初步探讨孕烷X受体(PXr)对黄曲霉毒素b1(Afb1)诱导肝细胞dnA损伤和坏死性凋亡的影响。方法采用已构建的PXr高表达l02-PXr和空白载体对照l02-P b细胞;实时荧光定量PCr(Q rT-PCr)检测细胞nr1I2和CyP3A4 M rnA水平改变;蛋白免疫印迹(WESTErn blOTTIng)检测细胞内PXr和坏死性凋亡下游效应自噬分子lC3-Ⅰ和lC3-Ⅱ蛋白相对表达含量;双核微核试验(CbMn)检测细胞遗传损伤情况;采用噻唑蓝(MTT)法测定Afb1对细胞活性抑制影响;利用坏死性凋亡抑制剂nEC-1构建坏死性凋亡抑制的细胞模型,验证Afb1诱导的坏死性凋亡的效应。结果与l02-P b细胞相比,l02-PXr细胞nr1I2 M rnA和PXr蛋白显著上调(均P<0.001)。Afb1显著地诱导l02-P b和l02-PXr细胞中CyP3A4 M rnA上调(均P<0.05),在l02-PXr细胞中的效应更为明显。与对照组相比,Afb1在5~30μMOl/l呈剂量反应关系诱导l02-P b和l02-PXr细胞的微核率增高(均P<0.05),l02-PXr细胞更为明显;同时,Afb1明显地诱导两株细胞的核芽率和核桥率,但随Afb1剂量增高都有下降趋势。细胞活性随Afb1浓度(1.875~120μMOl/l)增加呈剂量反应关系抑制(均P<0.05);且相对于l02-P b细胞,l02-PXr细胞对Afb1处理48 H诱导的细胞活性抑制作用更为敏感(P<0.05)。nEC-1可显著性抑制Afb1诱导的l02-PXr细胞活性抑制率(P<0.05),然而却不能降低Afb1诱导l02-P b细胞活性抑制率。此外,Afb1显著性诱导l02-P b和l02-PXr坏死性凋亡下游lC3-Ⅱ的上调(均P<0.05);且与l02-P b细胞相比,nEC-1对Afb1诱导的l02-PXr细胞活性抑制和lC3-Ⅱ上调的抑制效果更为明显(P<0.05)。结论 PXr参与Afb1诱导人肝细胞dnA损伤介导的坏死性凋亡,与PXr促进Afb1诱导CyP3A4基因上调有关。Objective To investigate the effects of pregnane X receptor(PXR) over expression on aflatoxin B1(AFB1)-induced DNA damage and necroptosis in human normal liver L02 cells.Methods The established cells models of stable transfection of over expression PXR(L02-PXR) and null vector p Babe-puro(L02-p B) were used.The background levels of NR1I2 m RNA and PXR protein, and the expression of AFB1-induced CYP3A4 m RNA and LC3-I / LC-3II protein were determined by the real time PCR(q RT-PCR) and Western blotting, respectively.The cytokinesis-block micronucleus(CBMN)assay was adopted to evaluate the genotoxicity.The cell viability inhibition rate was determined by MTT assay, after treatment with different doses of AFB1.The inhibition models of necroptosis were established by treatment with necroptosis inhibitor Nec-1.Results The expression of NR1I2 m RNA and PXR protein in L02-PXR cells were higher than that in L02-p B cells(all P<0.001).The level of CYP3A4 m RNA was significantly up regulated in L02-p B and L02-PXR cells by treatment with AFB1(all P<0.05).Compared with control group(Ctrl), MN frequencies in L02-p B and L02-PXR cells were significantly increased by treatment with AFB1 in a dose-dependent manner(all P <0.05), especially, in L02-PXR cells.Meanwhile, NBD and NBP frequencies were significantly increased by treatment with AFB1.However, AFB1 with a higher dose induced downward trends in frequencies of NBD and NBP.Moreover, the inhibition rate of cell viability was increased after treatment with AFB1(1.875~120 μmol / L) in a dose-dependent manner(all P <0.05); specifically, the inhibitory effects of AFB1-treatment after 48 h were significantly stronger in L02-PXR cells than in L02-p B cells(P <0.05).Interestingly, necroptosis inhibitor Nec-1 could inhibit AFB1-induced cell death in L02-PXR cells(P<0.05).On the contrary, Nec-1 could not prevent L02-p B cells from death by treatment with AFB1.In addition, the expression of necroptotic LC3-II, a classical marker of autophagy, was significantly increased by treatment with AFB1 in two cell lines(all P <0.05).Notably, pre-treatment with Nec-1 was able to block the inducement of necroptotic LC3-II in a more efficiently way in L02-PXR cells than in L02-p B cells(P <0.05).Conclusion PXR involved in the effect of AFB1 on necroptosis by DNA damage mediation in human liver cells;specifically, the up regulation of CYP3A4 gene may relate to the AFB1-induced DNA damage.国家自然科学基金(81172705;81072334); 广东省自然科学基金(S2011020002769); 福建省自然科学基金(2014J01372

Effects of cadmium on promoter methylation and transcriptional level of PPP2R1A gene in hepatocytes

目的分析镉染毒处理肝细胞中蛋白磷酸酶2A(PP2A)-Aα支架亚基基因PPP2r1A启动子区甲基化状态及其转录水平的改变。方法采用永生化人正常肝l02细胞及肝细胞癌HEP g2细胞为研究对象,对其进行以下分组和处理:1低、中和高剂量氯化镉(Cd Cl2)处理组,分别予浓度为20.0、40.0和60.0μMOl/l Cd Cl2处理24 H;2低、中和高剂量5-氮杂-2’-脱氧胞苷(5-AzA-d C)处理组,分别予浓度为2.5、5.0和10.0μMOl/l 5-AzA-d C处理48 H;3 5-AzA-d C组予浓度为5.0μMOl/l的5-AzA-d C处理48 H,Cd Cl2组予浓度为40.0μMOl/l的Cd Cl2处理24 H,(5-AzA-d C+Cd Cl2)组予浓度为5.0μMOl/l的5-AzA-d C预处理48 H后再予浓度为40.0μMOl/l Cd Cl2处理24 H;4 Cd Cl2处理组予浓度为40.0μMOl/l Cd Cl2处理24 H。上述4种分组均设对照组,予等体积生理氯化钠溶液或二甲基亚砜处理。经1~3处理后的细胞采用实时荧光定量聚合酶链反应(PCr)检测PPP2r1A、金属硫蛋白1b(MT1b)和dnA甲基转移酶3A(dnMT3A)的MrnA转录水平(以对照组水平为1.00)。经4处理后的细胞采用亚硫酸氢盐修饰后PCr扩增PPP2r1A启动子区克隆测序检测CP g岛的甲基化情况。结果 l02细胞和HEP g2细胞中,不同剂量Cd Cl2处理组PPP2r1A MrnA转录水平随镉处理剂量增高呈剂量依赖性下降(P0.05)。结论外源化学物Cd Cl2可诱导肝细胞中PPP2r1A转录水平降低,可能与镉能够引起目的基因启动子区甲基化状态改变有关,提示PP2A亚基基因的表观遗传学调控可影响镉诱导的肝细胞功能。Objective To analyze the effects of cadmium on the promoter methylation and transcriptional level of protein phosphatase 2A( PP2A)-Aα supported subunit gene PPP2R1 A gene in hepatocytes.Methods The immortalized human fetal liver cell line L02 and the hepatocellular carcinoma cell line Hep G2 were selected as the research objects: 1 Cells were treated with low-,medium- and high-dose( 20.0,40.0 and 60.0 μmol / L) cadmium chlorid( Cd Cl2) for 24 h.2Cells were treated with low-,medium- and high-dose( 2.5,5.0 and 10.0 μmol / L) 5-aza-2'-deoxycytidine( 5-Aza-d C)for 48 h.3 Cells were given 5.0 μmol / L for 48 h in 5-Aza-d C group,cells were exposed to 40.0 μmol / L Cd Cl2 for 24 h in Cd Cl2 group and cells were exposed to 40.0 μmol / L Cd Cl2 for 24 h after 48 h pretreatment of 5.0 μmol / L 5-Aza-d C in( 5-Aza-d C + Cd Cl2) group.4 Cells were treated with 40.0 μmol/L Cd Cl2 for 24 h in Cd Cl2 group.The above groups were all given the controls with the same volumes of physiological sodium chloride solution or dimethyl sulfoxide.Real-time fluorescent quantitative polymerase chain reaction( PCR) detection was used to detect the mRNA transcriptional levels of PPP2R1 A,Metallothionein 1B( MT1B),DNA methyltransferase 3A( DNMT3A) after treatments 1-3.After treatment4,cloning sequencing was used to detect the Cp G island methylation status of PPP2R1 A promoter after bisulfite sequencing PCR.Results In L02 and Hep G2 cells,the transcriptional levels of PPP2R1 A mRNA in Cd Cl2 group were decreased in a dose-dependent manner( P 0.05).Conclusion It was indicated the Cd Cl2 could lead to the transcription inhibition of PPP2R1 A,and the effect may be related with the change of its promoter methylation status.These data showed epigenetic regulation of PP2 A subunit genes may affect the function of hepatocytes exposed to cadmium.国家自然科学基金(81172705;81072334;81130052); 广东省自然科学基金重点项目(S2011020002769

CRISPR/Cas9敲低环氧合酶2表达对黄曲霉毒素B1诱导肝细胞核DNA损伤与脂质蓄积的影响

为探讨黄曲霉毒素B1(AFB1)诱导的肝细胞核DNA损伤与脂质蓄积的关系,以慢病毒质粒为载体,采用CRISPR/Cas9技术构建含靶向环氧合酶2(COX-2)编码基因(PTGS2)小向导RNA(sgRNA)的重组质粒,经测序验证成功后,制备假病毒感染HepG2细胞,构建稳定敲低COX-2表达的细胞.通过实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(WB)检测mRNA和蛋白水平,结果显示与HepG2-Cas9-NC对照细胞相比,HepG2-Cas9-PTGS2敲低细胞中PTGS2 mRNA与COX-2蛋白表达水平分别减少至(49.1±1.8)%和(48.1±0.7)%.给予细胞AFB1处理,通过WB和免疫荧光(IF)法检测DNA损伤标志物γH2AX的表达水平,结果显示处理组敲低细胞中,γH2AX蛋白水平和荧光焦点形成数目显著低于对照细胞(p<0.05);进一步检测脂质合成相关指标,发现敲低细胞中PPARγ蛋白、总胆固醇(TC)、总甘油三脂(TG)水平以及油红O染色阳性脂滴的分布密度,均显著低于对照细胞中(p<0.05).在敲低细胞中回补COX-2后给予AFB1处理,γH2AX蛋白水平和脂质分布密度均显著升高(p<0.05).综上,成功构建了HepG2-Cas9-PTGS2敲低细胞,并发现敲低COX-2表达对AFB1诱导的肝细胞核DNA损伤和脂质蓄积有显著抑制作用,为进一步研究靶向干预COX-2在外源化学物诱导肝细胞毒性中的作用机制提供了细胞模型和依据.国家自然科学基金(81573181,81472997,81773465);;福建省自然科学基金(2015J01344