【作者】 李荣；

【导师】 王取南；

【作者基本信息】 安徽医科大学 ， 卫生毒理学， 2021， 硕士

【摘要】 背景:邻苯二甲酸二（2-乙基己基）酯[di-（2-ethylhexyl）phthalate,DEHP]是一种广泛暴露的内分泌干扰物,可以破坏孕妇和胎儿的甲状腺激素调节系统,具有干扰胎盘滋养层细胞甲状腺激素（THs）转运的作用,其进入人体后可能主要通过邻苯二甲酸（2-乙基己基）单酯[mono-2-ethylhexyl phthalate,MEHP]发挥毒性作用。出生前胚胎发育所需的甲状腺激素主要由母体经胎盘转运来供给。而甲状腺激素对生物体神经系统的生长发育至关重要,在神经干细胞的增殖、分化、迁移中,都有甲状腺激素的调节作用。本研究拟观察MEHP对胚胎神经干细胞分化的影响。研究目的:探讨经MEHP处理的胎盘滋养层细胞对共培养胚胎神经干细胞分化的影响。方法:人胎盘滋养层细胞HTR-8经MEHP（0μM、50μM、100μM、180μM）处理24小时后,嵌入transwell共培养体系与人胚胎神经干细胞Re Ncell-CX（细胞融汇约70%）共培养72小时。嵌入前,收集MEHP处理HTR-8细胞0和24小时的培养液,用高效液相色谱-串联质谱法（LC-MS）检测MEHP含量;用细胞增殖-毒性检测试剂盒（CCK8）检测MEHP处理24小时的HTR-8细胞活力;用酶联免疫吸附试验（ELISA）检测MEHP处理HTR-8细胞0和24小时的培养液和细胞内三碘甲状腺素原氨酸（T₃）浓度,q RT-PCR和蛋白免疫印迹（WB）检测甲状腺激素代谢酶（DIO2、DIO3）以及受体（TRα、TRβ）的m RNA和蛋白水平。嵌入后,采用1%活性炭吸附处理血清（被认为不含甲状腺激素）的干细胞分化培养液培养（简称分化I液）,共培养72小时后,ELISA检测培养液中T₃含量,免疫荧光细胞化学法（ICC）和WB检测Re Ncell-CX细胞特异性抗体SOX2,IFC标记星形胶质细胞标志蛋白GFAP,WB检测神经元细胞标志β-TubulinⅢ。此外,神经干细胞分化实验中,设置一个1%未经活性炭吸附处理血清的分化培养液（简称分化II液）的非共培养阳性对照组,和一个分化I液培养的非共培养阴性对照。结果:（1）LC-MS检测结果显示培养液中MEHP含量与设计剂量一致。（2）最高剂量MEHP（180μM）处理组HTR-8细胞增殖率为88%,说明设计剂量未明显抑制细胞增殖,也未产生明显的细胞毒性。（3）MEHP处理减少了HTR-8细胞培养液中T3的消耗（P<0.01）。（4）MEHP处理引起HTR-8细胞甲状腺激素代谢酶DIO2的m RNA表达和蛋白水平下降,而DIO3的m RNA表达和蛋白水平升高（P<0.05）。（5）MEHP处理引起HTR-8细胞甲状腺激素受体（TRα、TRβ）的m RNA表达和蛋白水平下调（P<0.05）。（6）共培养后,神经干细胞向神经元方向分化减少（P<0.05）,向星形胶质细胞方向分化增多（P<0.05）。结论MEHP会影响人胎盘滋养层细胞HTR-8甲状腺激素脱碘酶和甲状腺激素受体的表达,处理后的HTR-8细胞会使共培养的人神经干细胞Re Ncell-CX向神经元方向分化减少,向星形胶质细胞方向分化增多。更多还原

【Abstract】 Background Di-(2-ethylhexyl)phthalate [di-(2-ethylhexyl)phthalate,DEHP] is a widely exposed endocrine disruptor that can damage the thyroid hormone regulation system of pregnant women and fetuses,and can interfere with placental nourishment The transport of thyroid hormones(THs)in layered cells may be toxic mainly through phthalate(2-ethylhexyl)monoester [mono-2-ethylhexyl phthalate,MEHP] after entering the human body.Thyroid hormones needed for embryonic development before birth are mainly supplied by the mother’s transport through the placenta.This study intends to observe the effect of MEHP on the differentiation of embryonic neural stem cells.Objective To explore the effect of MEHP-treated placental trophoblast cells on the differentiation of embryonic neural stem cells.Methods Human placental trophoblast cells HTR-8 were treated with MEHP(0μM,50μM,100μM,180μM)for 24 hours,and then embedded in a transwell co-culture system and human embryonic neural stem cells Re Ncell-CX(cell confluence about70%)were co-cultured for 72 hours.Before embedding,collect MEHP-treated HTR-8cell culture medium for 0 and 24 hours,use high performance liquid chromatography-tandem mass spectrometry(LC-MS)to detect MEHP content;use cell proliferation-toxicity detection kit(CCK8)to detect MEHP treatment Cell viability of HTR-8 cells for 24 hours;enzyme-linked immunosorbent assay(ELISA)was used to detect the 0 and 24 hours culture medium and intracellular triiodothyronine(T3)concentration of HTR-8 cells treated with MEHP,q RT-PCR and Western blotting(WB)detect the m RNA and protein levels of thyroid hormone metabolism enzymes(DIO2,DIO3)and receptors(TRα,TRβ).After embedding,use 1% activated charcoal adsorption treatment serum(think not containing thyroid hormone)stem cell differentiation culture medium(referred to as differentiation I liquid),after 72 hours of co-cultivation,ELISA test the content of T3 in the medium,immunofluorescence cytochemistry(IFC)and WB detect Re Ncell-CX cell-specific antibody SOX2,IFC labeled astrocyte marker protein GFAP,WB detects neuronal cell marker β-TubulinⅢ.In addition,in the neural stem cell differentiation experiment,a non-co-cultivation positive control group of differentiation medium(differentiation II for short)with 1%serum without activated carbon adsorption treatment was set up,and a non-co-culture negative control cultured in differentiation I medium.Results(1)The LC-MS test results showed that the MEHP content in the culture medium was consistent with the designed dose.(2)The proliferation rate of HTR-8cells in the highest dose MEHP(180μM)treatment group was 88%,indicating that the designed dose did not significantly inhibit cell proliferation and did not produce significant cytotoxicity.(3)MEHP treatment reduced the consumption of T3 in HTR-8cell culture medium(P<0.01).(4)MEHP treatment caused a decrease in the m RNA expression and protein level of thyroid hormone metabolizing enzyme DIO2 in HTR-8cells,while the m RNA expression and protein level of DIO3 increased(P<0.05).(5)MEHP treatment caused a down-regulation of m RNA expression and protein levels of thyroid hormone receptors(TRα,TRβ)in HTR-8 cells(P<0.05).(6)After co-cultivation,the differentiation of neural stem cells toward neurons decreased(P<0.05),and the differentiation toward astrocytes increased(P<0.05).Conclusions MEHP can affect the expression of HTR-8 thyroid hormone deiodinase and thyroid hormone receptor in human placental trophoblast cells.The treated HTR-8cells will reduce the differentiation of co-cultured human neural stem cells Re Ncell-CX towards neurons.The differentiation of astrocytes increased.更多还原