Luo Ling-feng, Yang Lin-qing, Wu De-sheng, Zhou Ming, Gong Chun-mei, Liu Qing-cheng, Xia Bo, Huang Guan-qin, Kuang Xia-feng, Zhuang Zhi-xiong, Zhang Wen-chang
Department of Occupational and Environmental Health, Fujian Medical University, Fuzhou, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2013 Feb;47(2):164-9.
To explore the effects of bisphenol A (BPA) exposure on toxicity characteristic and OCT4 and SOX2 gene expression of mouse embryonic stem cells (mESC).
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 (CCK8) 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.
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 effected 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) mol/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) mol/L (1.374 ± 0.053) dose 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 < 0.05). Moreover, the OCT4 protein expression level in the 10(-5) mol/L (1.360 ± 0.168) and 10(-4) mol/L (1.602 ± 0.151) were all significantly higher than the control group (1.000 ± 0.000) (t values were -3.538, -4.002 respectively, P value all < 0.05), while no obvious change of the SOX2 protein expression level was detected in all treated groups.
BPA in a certain dose range could upregulate the expression of OCT4 gene in mouse embryonic stem cells while had no significant effect on the expression of SOX2 gene.
探讨双酚A（BPA）暴露对小鼠胚胎干细胞（mESC）毒性特征及OCT4和SOX2基因表达的影响。
培养mESC，分别用10⁻⁸、10⁻⁷、10⁻⁶、10⁻⁵、10⁻⁴ mol/L的BPA及二甲基亚砜（DMSO，溶剂对照组）处理24小时，共处理三组细胞。通过倒置显微镜观察对照组和暴露组mESC的形态变化。采用细胞计数试剂盒8（CCK8）检测BPA对mESC增殖的影响，并据此计算半数抑制浓度（IC50）。运用实时荧光定量聚合酶链反应（RT-QPCR）和蛋白质免疫印迹法检测OCT4和SOX2的表达。
BPA对mESC具有一定毒性，BPA处理显著增加细胞毒性，呈浓度依赖性，IC50为4.3×10⁻⁴ mol/L，结合环境中BPA暴露浓度及相关文献，最终选取10⁻⁸、10⁻⁷、10⁻⁶、10⁻⁵、10⁻⁴ mol/L这五个浓度作为实验组。BPA处理24小时后mESC形态受到影响，与对照组相比，细胞数量减少，出现一些漂浮细胞，且在较高浓度组中细胞克隆变得不规则并发生分化。10⁻⁷ mol/L（1.146±0.087）、10⁻⁶ mol/L（1.156±0.030）、10⁻⁵ mol/L（1.158±0.103）和10⁻⁴ mol/L（1.374±0.053）剂量组的OCT4 mRNA表达水平均显著高于对照组（1.000±0.000）（t值分别为-2.384、-2.953、-3.203、-4.021，P值均<0.05）。同时，10⁻⁴ mol/L（1.113±0.052）剂量组的SOX2 mRNA表达水平高于对照组（1.000±0.000）（t值为-2.765，P值<0.05）。此外，10⁻⁵ mol/L（1.360±0.168）和10⁻⁴ mol/L（1.602±0.151）剂量组的OCT4蛋白表达水平均显著高于对照组（1.000±0.000）（t值分别为-3.538、-4.002，P值均<0.05），而所有处理组的SOX2蛋白表达水平均未检测到明显变化。
一定剂量范围内的BPA可上调小鼠胚胎干细胞中OCT4基因的表达，而对SOX2基因表达无显著影响。
