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Effect of Deoxynivalenol on Toxicity of Mouse Embryonic Stem Cells

Author: WuPing
Tutor: YuAiLian；JinKunLin
School: Taishan Medical College
Course: Pathogen Biology
Keywords: deoxynivalenol mouse Embryonic Stem cell cell proliferation cell cycle gene expression toxicity
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 32
Quote: 3
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Abstract


ObjectiveTo determine the effect of DON at the different concentration on mouse Embryonic Stem cells proliferation and cell cycle; and futher study the expression changes of SSEA-1, Nanog, Oct-4, Sox1 and Casp-3; the impact of the Notch signaling pwathway. For screening of pathogens and other toxins on the stem cell proliferation, the mechanism of embryonic development gene expression provide a theoretical basis.MethodsCultured mouse embryonic stem cells (mESC) were treated with different concentrations of DON for 24 hours. MTT assay and flowcytometry was performed to determine the proliferative and toxic effects of DON on mESC. Immunocytochemistry and RT-PCR were performed to determine the expression level of SSEA、Nanog、Oct-4、Sox1 and Casp-3, Finally, Western blot was performed to determine the activation of Notch signaling using anti-NICD、Jag1、Hes1 antibodies.Results1.effect of proliferation on stem cells: The MTT assay shows that compared with control group, the absorbance were decrease obviously, when mESCs were treated with DON at 50ng/ml and 100ng/ml, cell proliferation and survival rate were signicantly reduced, compared with vehicle treated group, especially when DON≥500ng/ml (P<0.01). When DON at 2000ng/ml, mES cells can be seen that the cell clone group and the density decreased more significantly than 500ng/ml, 1000ng/ml group.And the inhibitory effects of DON on proliferation was gradually enhanced with the increasing drugs concentration .2.effect on cell cycle: The results of Flow Cytometry demonstrated that after treated with 500ng/ml DON for 24h, number at S-phage were decrease significantly, compared with control group and 5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml group(P<0.01). 2000 ng/ml DON group the S-phase decreased significantly, but no significant difference between 500ng/ml group(P>0.05). The proliferation exponent of mESC was decreased distinctly with the increasing concentration of DON.3.impact on gene expression: Immunocytochemistry result showed that the average absorbance of mESC treated with 500ng/ml DON for 24h Oct4、Sox1、Nanog was increase apparently than control group’s. With the same treated time, the average absorbance of SSEA-1 and absorbance of Casp-3 were increase gradually along with DON concentration increase.The results of RT-PCR showed that the Oct-4、Nanog expression were inhibited after 500ng/ml DON treatment for 24 hours. There were statistical differences between experimental group and comparative group(P<0.01). With the increasing concentration of DON, the expression of Oct-4、Nanog were reduced. But there was no statistical differences between 1000ng/ml group and 500ng/ml group(P>0.05).4. the impact of the Notch signaling pathway: Western blots show that NICD, an activated form of Notch1, was signifincatly reduced when mESCs was treated at low concentration of DON(10 ng/ml). In addition, correspondingly, Jag1, a Notch1 receptor was also reduced gradually with the increasing concentration of DON. Moreover, Hes1, a downstream of Notch1 signaling, was also reduced, suggesting that Notch signaling was inhibited.Conclusion1.DON has toxic effects on mESC and can inhibit mESC growth, blocking cell to enter S phase.DON has a significant anti-proliferative effects.2.SSEA-1、Oct-4、Nanog is mES cell specific genes, which directly affect the transcription of mES cell differentiation and development. DON treatment could up regulate expression of SSEA-1、Casp-3 or down regulate the expression of Sox1、Oct-4、Nanog, thus affect mES cells proliferation and differentiation. Abnormal differentiation of can be damaged the normal development of early embryos.3.Notch signaling pathway have played an important role in maintaining stem cell properties, cell differentiation, proliferation, apoptosis, and so on. DON at low concentration(10ng/ml) can inhibit Notch signaling pathway, affect the characteristics of mES cells, proliferation and differentiation.

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