| Bisphenol A （bisphenol A, BPA） is widely present in the living environment ofhuman, it is closely related with our various production and living activities, we aregenerally exposed to bisphenol A, especially the exposure of the pregnant womenbecomes a growing concern.The classic toxicological studies pointed out that a certaindose of BPA may inhibit the embryonic development. Before the end of the19thcentury, due to the limitations of the experimental techniques and medical ethics, it isdifficult to carry out early embryonic development in utero, whether BPA effets earlyembryo development and the possible mechanisms are poorly charaeterized andremain in the stage of animal experiments. Since the rapid development of the theoryand technology of embryonic stem cells and its applications in the end of the20thcentury, it provides a new means for the toxicity studies of exogenous chemicals. Inthis study,the mESC were treated with BPA,and a series of related vitro experimentswere launched to evaluate embryo toxicity of bisphenol A at the cellular level, toprovide a scientific basis for the rational evaluation of BPA safety; moreover, weobserved whether BPA can change the level of gene promoter methylation to affectthe expression of specific genes, to provide new ideas for further study of the affect ofBPA in early embryonic development and its mechanisms.Objectives:1、S6mouse embryonic stem cells were cultured by in vitro feeder layer.Embryonic stem cell experiments （EST） were used to evaluate the embryotoxiceffects of BPA, and observing effects of bisphenol A on the growth anddifferentiation of EBs, to provide a scientific basis for the rational evaluation of BPAsafety and exploring the effects of bisphenol A on mouse embryonic stem cells andtheir differentiation potential.2、mES cells were cultured with conditioned medium,cytotoxicity and gene expresssion of OCT4, SOX2and Nanog of the different concentrations of BPA were obeserved and DNA methylation in OCT4gene promoterregion of the MES cells with BPA were detected, to further study effects of BPA onearly embryonic development and its mechanism.Methods：1、 Cultured mES cells supported by a feeder layer, the solvent control（DMSO）, BPA (10-7mol/L,10-6mol/L,10-5mol/L,10-4mol/L,10-3mol/L, theconcentration of BPA covers the control of environmentally relevant concentrationsand higher concentrations) were set. Using the hanging drop, suspension culture,detecting the ability of MES cells in vitro differentiating into myocardial cells underdifferent concentrations of BPA,morphological changes were observed under aninverted microscope. Combined with the corresponding CCK-8 to detect50%inhibitory concentration (IC50) of MES cells and mice embryonic fibroblast3T3treated with BPA, differentiation and inhibition test was conducted to get50%differentiation inhibitory concentration （ID50） of mouse embryonic stem cells treatedwith bisphenol A. According to the discriminant formula embryo toxicity of bisphenolA was evaluated to select the appropriate concentration for post-induction ofdifferentiation.2、 Referring to half of the ES cell differentiation inhibitoryconcentration and the relevant literature, the solvent control （DMSO）, BPA (10-9mol/L,10-8mol/L,10-7mol/L,10-6mol/L) and E210-9mol/Lestradiol control groupwere set. MES cells were cultured by the hanging drop and suspension to obtainembryo body （EB）, and without any inducing factor were cultured for10d.Real-time fluorescence quantitative PCR （RT-Q-PCR） method were used to detectmRNA expression of the typical entoderm gene （Transthyretin, AFP）,typicalmesoderm gene （Flk-1, GATA-1）, typical ectoderm gene （Vimentin, Nestin） in theEB, observing effects of bisphenol A on the growth and differentiation of EBs.3、The solvent control （DMSO）, BPA (1.56×10-5 mol/L,3.12×10-5 mol/L,6.25×10-5 mol/L,1.25×10-4 mol/L,2.5×10-4mol/L,5×10-4 mol/L,1×10-3 mol/L,2×10-3 mol/L were set. mES cells were exposed for24h. CCK-8 method wasused to detect the effect of cell proliferation to caculate IC50. mES cells were exposedto different concentrations of BPA (0,10-8 mol/L,10-7 mol/L,10-6 mol/L,10-5mol/L,10-4 mol/L) for24h, real-time PCR and Western blot were used to detect the OCT4, SOX2expression at the mRNA and protein levels by bisphenol A.4、Combined with base-specific cleavage reaction with MALDI-TOF-MS detectionprinciple, the Sequenom’s MassARRAY inspection system detected the DNAmethylation of the OCT4gene promoter region.Results:1、According to EST, three reaction endpoint IC50ES, IC503T3, ID50,:5.22×10-4mol/L,6.25x10-4mol/L,7×10-7mol/L, respectively, were obtainedinto three linear discriminant function of the prediction model. Acording to the modelcriteria, BPA is a srong embryotoxic compound.2、In the certain concentration range,BPA has no statistical effects on typical entoderm gene （Transthyretin AFP） andtypical ectoderm genes （Vimentin, Nestin）（P>0.05）; while the typical mesodermgene （Flk-1, Gata-1） increased at lower concentrations of10-9mol/L and10-8mol/L and then decreased at a high concentration group and positive control group（P<0.05）.3、 BPA has certain toxic effects on mESC. The greater the exposureconcentration, the smaller the cell activity. They showed a dose-responserelationship. IC50 of BPA on mESC is4.3×10-4 mol/L. Refering to BPA exposureand related information,10-8、10-7、10-6、10-5、10-4 mol/L were used for the furtherexperiment. Treated with BPA for24h, mESC cell morphology changed. Comparedwith control, the higher dose group showed the number of cells was significantlyreduced, a little floating cells came out and cloning became irregular anddifferentiation became evident. OCT4mRNA relative expression levels were higherat the BPA concentration of10-7mol/L,10-6mo/L,10-5 mol/L,10-4mol/L（p<0.05）. SOX2mRNA relative expression levels were higher than control group atthe concentration of10-4mol/L（P <0.05）. Nanog mRNA levels were lower thanthose in the control group at the concentration of10-6 mol/L,10-5 mol/L,10-4 mol/L（P <0.05）. OCT4protein relative expression levels were higher than the DMSOgroup at the concentration of10-5 mol/L,10-4 mol/L（P <0.05）. SOX2proteinexpression levels were not found to change significantly at any concentration. Nanogprotein expression level was first increased to the top at the concentration of10-7mol/L and then decreased to the nadir at the concentration of10-4mol/L（P <0.05）.4、In SssI methyl transferase enzyme-treated DNA samples, CpG methylationlevel was significantly higher than any other group of DNA samples （P <0.05）,indicating good sensitivity of the detection method. Compared with the solventcontrol group, OCT4gene CpG methylation rate in DAC and TSA treatment groupwas significantly lower（P <0.05）; compared with the solvent control group, CpGmethylation rate treated with BPA showed a downward trend with the increaseddose,but the change is not significant. Only at the concentration of10-5 mol/L thedecrease was statistically significant （P <0.05）.Conclusions:1、 The C57/BL6mouse-derived mouse embryonic fibroblasts were successfullyisolated and cultured and could be a subculture within the7th generation. MES cellswere successfully cultured. The mouse EST system was initially established and BPAembryo toxic effects were classified to conclude that BPA is the strong embryo toxiccompound.2、 mES cells successfully differentiated into embryoid bodies. In a certainconcentration range, with increasing concentration of BPA exposure, the embryomesoderm characteristic gene expression first increased and then decreased,indicating that in respectively lower concentration BPA had infulence ondifferentiation potential of mES cells, especially promoting to mesoderm.3、 BPA can enhance the OCT4gene expression within a certain range ofconcentration. It had no significant effect on SOX2gene expression while it reducedthe expression of Nanog gene to some extent, indicating that OCT4, SOX2and Nanoggene coorperated to maitain the ES cell pluripotencyand self-renewal capacity andearly embryonic development jointly.4、In the role of BPA on mES cell processes, OCT4gene promoter region showedhypomethylation. The methylation status did not change significantly at lowconcentrations, while it decreased only at higher concentrations. |
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