α-生育酚对亚硝胺诱导人食管上皮细胞癌变早期NFκB和Nrf2信号通路的调节作用  

Regulation of α-tocopherol on NFKB and Nrf2 signaling pathway at early stage of N-nitrosomethylbenzylamine-induced human esophageal cell carcinogenesis

作　　者：杨辉[1] 孙拿拿[1] 李永宁[1] 梁春来[1] 贾旭东[1] 

机构地区：[1]卫生部食品安全风险评估重点实验室 国家食品安全风险评估中心,北京100021

出　　处：《中华预防医学杂志》2015年第6期546-553,共8页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金（81402679）;国家食品安全风险评估中心青年科研基金（2014007）

摘　　要：目的研究α-生育酚（d—tocopherol，α-T）对亚硝胺诱导人食管上皮细胞癌变早期NFκB和Nrf2信号通路的调节作用。方法采用N-甲基苄基亚硝胺（N—nitrosomethylbenzylamine，NMBzA）染毒人正常食管上皮细胞（HET-1A）模拟食管癌变启动，染毒浓度为50、100μmol／L，染毒时间为24h。α-T干预组细胞在染毒前3h加入25、50、100μmol／L的仅一T进行预处理，然后与100μmol／LNMBzA共同孵育24h；人食管鳞癌细胞（EC109）细胞采用相同浓度的α-T进行处理并与HET-1A进行对比；以体积分数为0．1％二甲基亚砜（DMSO）处理组细胞作为对照组。采用细胞免疫荧光试验分析NFκB p65和Nrf2在细胞内的分布和活化状态，采用RT-PCR或Western blot法检测细胞周期素D1（cyclinD1）、核蛋白K167（K167）、增殖细胞核抗原（PCNA）、环氧化酶2（COX2）、5脂氧合酶（5LOX）、血红素加氧酶1（HO-1）、醌氧化还原酶（NQO1）、谷胺酸半胱氨酸连接酶（GCLC）等相关靶基因表达水平，采用流式细胞术检测细胞内活性氧水平。结果HET-1A细胞经100μmol／LNMBzA染毒后，CyclinD1、K167、PCNA基因表达水平分别为2．99±0．15、2．35±0．38、2．46±0．25，均高于对照组（1．00±0．08）（F值分别为97．23、65．28、34．62，P值均〈0．001）；与对照组（0）相比，NFκB p65（71．0％，98／138）和Nrf2（36．3％，49／135）在细胞核内的分布比例增加（χ^2值分别为194．71、133．72，P值均〈0．001）。与对照组（1．00±0．05）相比，HET-1A靶基因COX2（3．22±0．17）、5LOX（2．87±0．12）和HO-1（1．87±0．22）、NQ01（2．14±0．08）、GCLC（2．63±0．41）蛋白表达水平均升高（F值分别为72．35、43．87、69．23、71．34、85．79，P值分别为0．013、0．015、0．010、0．011、0．002）。50μmol／L α—T干预处理后，与100μmol／L NMBzA组（71．0％，98／138）相比，NFκB p65在核内分�Objective To investigate the regulation of α-Tocopherol on NFκB and Nrf2 signaling pathway at early stage of N-nitrosomethylbenzylamine （NMBzA）-induced human esophageal carcinogenesis. Methods Human normal esophageal HET-1A cells were treated with NMBzA at 50 μmol/L, 100 μmol/L for 24 h to intimate the initiation of esophageal carcinogenesis. For intervention groups, HET- 1A cells were pre-treated with α-T at 25, 50, 100 μmol/L for 3 h and then co-treated with NMBzA （100 μmol/L） for 24 h. In comparison with HET-1A cells, human esophageal cancer EC109 cells were treated with α-T at corresponding concentrations. Cells treated with 0.1% DMSO were used as negative control.Immunofluorence staining was used for the determination of distribution and activation of NFκB p65 and Nrf2 in the cell. Real time PCR and Western blot were used to determine the expression levels of target genes including cyclinD1, KI67, proliferating cell nuclear antigen（PCNA）, cyclo-oxygen-ase 2（COX2）, 5LOX, HO-1, NQO1 and GCLC. Flow cytometry was utilized to analyze the reactive oxygen species contents in the cells. Results As compared to the control group （1.00±0.08）, the expression of CyclinD1 （2.99±0.15）, KI67 （2.35 ± 0.38） and PCNA （2.46± 0.25） in HET-1A were all markedly increased by NMBzA treatment （F values were 97.23,65.28,34.62,P〈0.001）. Also, the proportion of cells with nucleus translocation of NFκB p65 （71.0%,98/138） or Nrf2 （36.3%,49/135） were significantly increased（χ^2 values were 194.71,133.72,P〈0.001）, and the expression of COX2 （3.22±0.17）, 5LOX （2.87±0.12） as well as HO- 1 （1.87±0.22）, NQO1 （2.14±0.08）, GCLC （2.63±0.41） at protein levels were elevated （F values were 72.35, 43.87, 69.23, 71.34, 85.79,P values were 0.013, 0.015, 0.010, 0.011, 0.002）. Under the treatment with 50 μmol/L α-T, comparing with the control group（59.1%,65/110）,the nuclear translocation of NFκB p65 （77.7%,8/104） was clearly inhibited （χ^2=148.1,

关 键 词：Α-生育酚 食管肿瘤 NFKB NRF2 活性氧自由基 

分 类 号：R735.1[医药卫生—肿瘤]