【作者】 李岩；

【导师】 董静；

【作者基本信息】 中国医科大学 ， 劳动卫生与环境卫生学， 2023， 硕士

【摘要】 目的:邻苯二甲酸二(2-乙基己基)酯(di-(2-ethylhexyl)phthalate,DEHP),它是一种广泛使用的增塑剂,且被认为是一种潜在的环境内分泌干扰物,并会引起一些神经退行性行为。然而,DEHP及其代谢物邻苯二甲酸单(2-乙基己基)酯(mono(2-ethylhexyl)phthalate,MEHP)对脑皮质发育的影响尚不完全清楚,有待进一步的探讨。本研究拟通过构建DEHP暴露动物模型探讨DEHP是否会对胎鼠皮质的发育造成影响,以及MEHP单独或联合络丝蛋白(Reelin)特异性干预剂处理大鼠肾上腺嗜铬细胞瘤细胞(Pheochromocytoma-derived cell,PC12)对Reelin信号通路的影响。方法:用SPF级成年Wistar大鼠构建DEHP暴露动物模型,从妊娠当天(encyesis0,E0)直至妊娠第21天(E21),用0 mg/kg/day,300mg/kg/day DEHP浓度玉米油对大鼠进行灌胃给药。在E17,E19和E20三个时间点分别进行取材,对胎鼠大脑皮质进行包埋切片和H&E(Hematoxylin-eosin staing)染色、Nissl染色和免疫荧光染色、PCR(Quantitativa Real-time PCR,q RT-PCR)实验、Elisa实验和Western-Blot实验分别检测大脑皮质神经细胞增殖,细胞周期退出,神经元细胞的径向迁移;Reelin信号通路Reelin、载脂蛋白E受体2(Apolipoprotein E receptor,-2,Apo ER2)、极低密度脂蛋白受体(Very low density lipoprotein receptor,VLDLR)和Disabled-1蛋白(Disabled,Dab1)、以及其下游的Crk和Nectin3的m RNA以及蛋白表达含量的变化情况。体外实验以PC12细胞为研究对象,采用细胞计数试剂(Cell Counting Kit-8,CCK-8)法检测细胞活力,并建立体外MEHP神经毒性的细胞模型。通过分别给予Reelin特异性激动剂褪黑素(Melatonin,MT)和Reelin蛋白特异性抑制剂链脲佐菌素(Streptozotocin,STZ)处理,建立其与MEHP共暴露模型,采用实时荧光定量PCR法检测RELN、Apo ER2、VLDLR、Dab1、Crk和Nectin3的m RNA表达水平;采用Elisa法检测Reelin蛋白的表达水平;采用Western-Blot法检测Dab1、p-Dab1、Crk和Nectin3的蛋白表达含量。并采用划痕实验观察MEHP暴露和MT干预以及MEHP和MT共暴露对PC12细胞迁移能力的影响。结果:(1)在E20胎鼠脑皮质中,与对照组相比,DEHP暴露组的大鼠脑皮质矢状面切片H&E和Nissl染色未见组织病理学改变异常。在E17和E19天的胎鼠脑皮质中,与对照组相比,DEHP暴露组心室下区-心室区(subventricular zone-ventricular zone,SVZ-VZ)中的ki67~+细胞数量和Brd U~+细胞数量减少,差异具有统计学意义(p<0.05);并且可以观察到细胞增殖随着年龄的增长而下降。E17天胎鼠脑皮质计算细胞周期退出指数,与对照组相比,DEHP暴露会导致细胞退出周期指数增加,差异具有统计学意义(p<0.05);在E20的胎鼠脑皮质中,与对照组相比,DEHP暴露组分布于SVZ-VZ层的Brd U~+细胞比例高于对照组,说明DEHP暴露导致向皮质板迁移的细胞数量减少,而在中间层(intermediate zone,IZ),DEHP暴露组Brd U~+细胞的比例低于对照组,说明细胞迁移发生了延迟,差异具有统计学意义(p<0.05)。(2)与对照组相比,DEHP暴露组RELN、Apo ER2、VLDLR、Dab1、Crk和Nectin3的m RNA表达水平降低,差异具有统计学意义(p<0.05)。与对照组相比,DEHP暴露组Reelin、Dab1、p-Dab1、Crk和Nectin3的蛋白水平均降低,差异具有统计学意义(p<0.05)。(3)在体外划痕实验中,MEHP暴露组的空白面积大于对照组,说明MEHP会使细胞迁移能力下降;MT组空白面积小于对照组,说明MT会使细胞迁移能力增强;MEHP+MT组空白面积小于MEHP组,说明MT的干预会使细胞迁移能力有所恢复。(4)与对照组相比,MEHP组RELN、Apo ER2、VLDLR、Dab1、Crk和Nectin3的m RNA表达均降低,差异具有统计学意义(p<0.05),MT组m RNA表达升高,差异具有统计学意义(p<0.05),STZ组m RNA表达降低,差异具有统计学意义(p<0.05)。与MT组相比,MEHP+MT组RELN、Apo ER2、VLDLR、Dab1、Crk和Nectin3的m RNA表达降低,差异具有统计学意义(p<0.05)。与STZ组相比,MEHP+STZ组RELN、Apo ER2、VLDLR、Dab1、Crk和Nectin3的m RNA表达降低,差异具有统计学意义(p<0.05)。与MEHP组相比,MEHP+MT组RELN、Apo ER2、VLDLR、Dab1、Crk和Nectin3的m RNA表达升高,差异具有统计学意义(p<0.05),MEHP+STZ组m RNA表达降低,差异具有统计学意义(p<0.05)。与对照组相比,MEHP组Reelin、Dab1、p-Dab1、Crk和Nectin3的蛋白表达水平降低,差异具有统计学意义(p<0.05),MT组蛋白表达水平升高,差异具有统计学意义(p<0.05),STZ组蛋白表达水平降低,差异具有统计学意义(p<0.05)。与MT组相比,MEHP+MT组Reelin、Dab1、p-Dab1、Crk和Nectin3的蛋白表达水平降低,差异具有统计学意义(p<0.05)。与STZ组相比,MEHP+STZ组Reelin、Dab1、p-Dab1、Crk和Nectin3的蛋白表达水平降低,差异具有统计学意义(p<0.05)。与MEHP组相比,MEHP+MT组Reelin、Dab1、p-Dab1、Crk和Nectin3的蛋白表达水平升高,差异具有统计学意义(p<0.05),MEHP+STZ组蛋白表达水平降低,差异具有统计学意义(p<0.05)。结论:(1)妊娠期DEHP暴露抑制胎鼠大脑皮质神经细胞增殖和细胞周期退出,导致细胞迁移障碍;(2)DEHP暴露抑制Reelin及其下游信号分子的表达。(3)胎鼠大脑皮质神经细胞增殖减少、细胞周期退出抑制以及细胞迁移障碍与Reelin信号通路有关。更多还原

【Abstract】 Objective:Di-(2-ethylhexyl)phthalate(DEHP)is a widely used plasticizer and is considered a potential environmental endocrine disruptor that can cause some neurodegenerative behaviors.However,the effects of DEHP and its metabolite mono(2-ethylhexyl)phthalate(MEHP)on cortical development are not fully understood and further exploration is needed.This study aims to explore whether DEHP can affect the development of fetal rat cortex by constructing an animal model exposed to DEHP,and the effect of MEHP alone or in combination with a specific intervention agent of REELIN on the REELIN signaling pathway in rat adrenal pheochromocytoma derived cell(PC12).Methods:A DEHP exposure animal model was constructed using SPF grade adult Wistar rats.From the day of pregnancy(Encyesis 0,E0)to the 21st day of pregnancy(E21),rats were gavaged with 0 mg/kg/day,300mg/kg/day DEHP concentration corn oil.The samples were taken at three time points of E17,E19 and E20,and the embryonic rat cerebral cortex was subjected to embedding section,H&E(Hematoxylin eosin staging)staining,Nissl staining and immunofluorescence staining,PCR(Quantitative Real time PCR,q RT PCR)experiment,Elisa experiment and Western Blot experiment to detect the proliferation,cell cycle exit and radial migration of neurons in the cerebral cortex respectively;Changes in m RNA and protein expression levels of the Reelin signaling pathway,Apolipoprotein E receptor 2(Apo ER2),Very low density lipoprotein receptor(VLDLR),Disabled 1 protein(Dab1),as well as downstream Crk and Nectin3.In vitro experiments were conducted on PC12 cells,using Cell Counting Kit 8(CCK-8)to detect cell viability and establish a cell model for MEHP neurotoxicity in vitro.A co exposure model with MEHP was established by treating with the Reelin specific agonist melatonin(MT)and the Reelin protein specific inhibitor Streptozotocin(STZ),respectively.Real time fluorescence quantitative PCR was used to detect the m RNA expression levels of RELN,Apo ER2,VLDLR,Dab1,Crk,and Nectin3;Using Elisa method to detect the expression level of Reelin protein;Western Blot method was used to detect the protein expression levels of Dab1,p-Dab1,Crk,and Nectin3.Scratch test was used to observe the effects of MEHP exposure,MT intervention and MEHP and MT co exposure on PC12cell migration.Results:(1)In the brain cortex of E20 fetal rats,compared with the control group,no abnormal histopathology changes were found in the sagittal plane section of the brain cortex of DEHP exposed rats by H&E and Nissl staining.Compared with the control group,the DEHP exposure group showed a statistically significant decrease in the number of ki67~+cells and Brd U~+cells in the subventricular zone ventricular zone(SVZ-VZ)of fetal rat cerebral cortex on days E17 and E19(p<0.05);And it can be observed that cell proliferation decreases with age.The cell cycle exit index was calculated in the cortex of E17 day fetal mice.Compared with the control group,DEHP exposure resulted in an increase in the cell cycle exit index,with a statistically significant difference(p<0.05);In the cortex of E20 fetal rats,compared with the control group,the proportion of Brd U~+cells distributed in the SVZ-VZ layer in the DEHP exposed group was higher than that in the control group,indicating that DEHP exposure led to a decrease in the number of cells migrating to the cortical plate,while in the intermediate zone(IZ),the proportion of Brd U~+cells in the DEHP exposed group was lower than that in the control group,indicating that cell migration was delayed,with a statistically significant difference(p<0.05).(2)Compared with the control group,the m RNA expression levels of RELN,Apo ER2,VLDLR,Dab1,Crk,and Nectin3 in the DEHP exposure group decreased significantly(p<0.05).Compared with the control group,the protein levels of Reelin,Dab1,p-Dab1,Crk,and Nectin3 in the DEHP exposure group decreased significantly(p<0.05).(3)In the scratch test in vitro,the blank area of MEHP exposed group was larger than that of the control group,indicating that MEHP would reduce the ability of cell migration;The blank area of MT group was smaller than that of control group,indicating that MT could enhance the ability of cell migration;The blank area of MEHP+MT group was smaller than that of MEHP group,indicating that MT intervention could restore the ability of cell migration.(4)Compared with the control group,the m RNA expression of RELN,Apo ER2,VLDLR,Dab1,Crk,and Nectin3 in the MEHP group decreased with statistical significance(p<0.05).The m RNA expression in the MT group increased with statistical significance(p<0.05),while the m RNA expression in the STZ group decreased with statistical significance(p<0.05).Compared with the MT group,the m RNA expression of RELN,Apo ER2,VLDLR,Dab1,Crk,and Nectin3 in the MEHP+MT group decreased significantly(p<0.05).Compared with the STZ group,the m RNA expression of RELN,Apo ER2,VLDLR,Dab1,Crk,and Nectin3 in the MEHP+STZ group decreased significantly(p<0.05).Compared with the MEHP group,the m RNA expression of RELN,Apo ER2,VLDLR,Dab1,Crk,and Nectin3 in the MEHP+MT group increased with statistical significance(p<0.05),while the m RNA expression in the MEHP+STZ group decreased with statistical significance(p<0.05).Compared with the control group,the protein expression levels of Reelin,Dab1,p-Dab1,Crk,and Nectin3 in the MEHP group decreased with statistical significance(p<0.05),the protein expression levels in the MT group increased with statistical significance(p<0.05),and the protein expression levels in the STZ group decreased with statistical significance(p<0.05).Compared with the MT group,the protein expression levels of Reelin,Dab1,p-Dab1,Crk,and Nectin3 in the MEHP+MT group decreased significantly(p<0.05).Compared with the STZ group,the protein expression levels of Reelin,Dab1,p-Dab1,Crk,and Nectin3in the MEHP+STZ group decreased significantly(p<0.05).Compared with the MEHP group,the protein expression levels of Reelin,Dab1,p-Dab1,Crk,and Nectin3 in the MEHP+MT group increased with statistical significance(p<0.05),while the protein expression levels in the MEHP+STZ group decreased with statistical significance(p<0.05).Conclusion:(1)DEHP exposure during pregnancy inhibits the proliferation and cell cycle withdrawal of neural cells in the cerebral cortex of fetal rats,leading to the obstacle of cell migration;(2)DEHP exposure inhibits the expression of Reelin and its downstream signaling molecules.(3)The decreased proliferation of neural cells in the cerebral cortex of fetal rats,the inhibition of cell cycle withdrawal and the obstacle of cell migration are related to the Reelin signaling pathway.更多还原