双酚A对小鼠胚胎干细胞OCT4及SOX2基因表达的影响  

Effects of bisphenol A on OCT4 and SOX2 genes expression in mouse embryonic stem cells

作　　者：罗凌凤[1] 杨淋清[2] 吴德生[2] 周明[2] 龚春梅[2] 刘庆成[2] 夏菠[2] 黄冠琴[2] 匡侠锋[2] 庄志雄[2] 张文昌[1] 

机构地区：[1]福建医科大学公共卫生学院职业与环境卫生学系,福州350108 [2]深圳市疾病预防控制中心现代毒理学重点实验室,卫生毒理医学重点实验室

出　　处：《中华预防医学杂志》2013年第2期164-169,共6页Chinese Journal of Preventive Medicine

基　　金：基金项目：国家自然科学基金（81172710）;深圳市科技研发资金基础研究计划（JC201105180762A）,深圳市科技计划项目（201202097）

摘　　要：目的探讨双酚A（bisphenolA，BPA）对小鼠胚胎干细胞（mESC）的毒性特征及OCT4、SOX2基因表达的影响。方法分别以10^-8、10^-7、10^-6、10^-5、10^-4mol／L浓度的BPA及二甲亚砜（溶剂对照组，DMSO）处理mESC系（S6）细胞24h，重复处理3次，倒置显微镜下观察正常组、染毒组细胞的形态变化，CCK-8法检测BPA对细胞增殖的影响，计算半数抑制浓度（IC50）；分别采用real—time（RT）一Q—PCR和western—blotting技术检测BPA作用下OCTg、SOX2基因在mRNA和蛋白水平表达的变化。结果BPA对mESC有一定的毒性作用，染毒浓度越大，细胞活性越小，二者呈剂量-效应关系，并通过计算得出BPA对mESC的IC50为4．3×10^-4mol／L，同时结合BPA的环境相关暴露浓度和相关资料，最终取10^-8、10^-7、10^-6、10^-5、10^-4mol／L系列浓度作为后续实验浓度；BPA处理24h后，mESC细胞形态发生改变，较高浓度组与对照组比较，细胞数量明显减少，出现少许漂浮的细胞，克隆变得不规则，分化趋势明显。BPA浓度为10^-7、10^-6、10^-5、10^-4的OCT4mRNA相对表达量分别为1．146±0．087、1．156±0．030、1．158±0．103、1．374±0．053，均高于对照组（1．000±0．000）（t值分别为-2．384、-2．953、-3．203、-4．021，P值均〈0．05）；BPA染毒浓度为10^-4mol／L（1．113±0．052）的SOX2mRNA相对表达水平高于对照组（1．000±0．000）（t=-2．765，P〈0．05）。染毒浓度为10^-5、10^-4mol／L的OCT4蛋白质相对表达量分别为1．360±0．168、1．602±0．151，均高于DMSO组（1．0004-0．000），差异有统计学意义（t值分别为-3．538、-4．002，P值均〈0．05）；而各浓度组SOX2蛋白相对表达水平差异无统计学意义。结论BPA对mESC具有一定的细胞毒性，在一定剂量范围内BPA可以使mESCOCT4基因表达增加，而对SOX2基因无明显影响。Objective To explore the effects of bisphenol A （BPA） exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells （mESC）. Methods mESC were cultured, and treated with the doses of 10 ^-8, 10^ -7,10 ^-6,10 ^-5,10 ^-4 mol/L respectively of BPA and DMSO （the solvent control group）for 24 hours, and three groups of cells were treated with the same method. The morphological changes of mESC in the control and exposure groups were observed through an inverted microscope. Cell counting kit 8 （CCKS） was used to detect the effects of BPA on proliferation of mESC, and based on the results ,the half inhibitory concentration（ IC50） was calculated. Real-time fluorescent quantitative polymerase chain reaction （RT-QPCR） and western blotting were used to detect the expression of OCT4 and SOX2. Results BPA had certain toxicity on mESC, the treatment of BPA significantly increased cell toxicity in a concentration-dependent manner, and the IC50 was 4. 3× 10^-4 mol/L, combined with the BPA exposure concentration of the environment and the related literature, eventually taking the five concentrations of 10^- 8,10 ^-7,10^-6,10 ^-5,10 ^-4 mol/L as the experimental groups. The mESC morphology were effeeted after the treatment of BPA for 24 h, compared with the control group, the number of cells decreased,appearing some floating cells, and the cell cloning became irregular and differentiation in the higher concentration groups. The OCT4 mRNA expression level in the 10^- 7 mo]/L （1. 146 ± 0. 087 ）, 10^-6 moL/L （1.156±0.030）,10^-5 mol/L （1.158±0. 103） and the 10-4 tooL/L（1.374 ±0.053） close group were all significantly higher than the control group （1. 000 ± 0. 000 ） （t values were -2. 384, - 2. 953, - 3. 203, - 4. 021 respectively,P value all 〈 0. 05 ）. Meanwhile,the SOX2 mRNA expression level in the 10 4 mol/L（ 1.113 ± 0. 052）were higher than the control group （1. 000 ± 0. 000） （t value was -2. 765 ,P value 〈 O. 05 ）. Moreover

关 键 词：胚胎干细胞 小鼠 双酚A OCT4基因 SOX2基因 

分 类 号：R114[医药卫生—卫生毒理学]