【作者】 王婷婷；

【导师】 陈晓萍；

【作者基本信息】 浙江工业大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 有机磷农药地亚农(Diazinon,DZN)在低浓度下能产生胚胎神经发育毒性,造成胚胎神经前体细胞发育异常,进而导致成年后人或动物的认知行为和情绪反应异常,但是其毒性作用的具体机制还有待近一步研究。本文从顶端神经前体细胞(apical progenitor cells,APs)分裂进程着手,研究DZN对APs周期性核移行(Interkinetic nuclear migration,INM)过程的影响,分析所造成的增殖分化结局,并且评估了这种作用的持续性。本文第二章通过检测三种基本的酶活性,分析DZN对脑组织的急性毒性作用及其恢复情况。对妊娠14天的小鼠间隔3小时连续三次暴露0、1、5mg/kg DZN,分别于E14、E16、E18三个时间测定母鼠血的ACh E活力,胚胎脑的AChE活力、Caspase-3活力、SOD活力。结果显示,5mg/kg DZN暴露造成E14、E16、E18母鼠血胆碱酯酶活力分别降至正常值的40.25%、60.34%、80.32%;E14、E16胚胎脑胆碱酯酶活力分别降至正常值的62.53%和79.41%,在E18天恢复接近正常值;E14、E16胚胎脑Caspase-3活性分别升高至正常值的148.64%、113.86%,在E18天恢复接近正常值;三个时间点的SOD活性均无显著性变化。本文第三章研究DZN对神经前体细胞分裂增殖的影响。对染毒妊娠小鼠腹腔单次注射5-溴脱氧尿嘧啶核苷(5-bromo-2’-deoxyuridine,Brdu)以标记S期细胞,制备Brdu标记1h、3h、6h定时胚胎,进行Brdu免疫组化染色。结果显示,注射1mg/kg DZN后,1h与3h时Brdu标记核位置无明显变化,6h时Brdu标记核的位置较对照组滞后。5mg/kg DZN暴露后,1h、3h、6h时Brdu标记核的位置均较对照组滞后,即DZN减缓了APs的核移行运动。本文第四章研究DZN对神经前体细胞发育演变的影响。对染毒小鼠胚胎进行Pax6、Tbr2、Tbr1免疫组化染色,结果显示5mg/kg DZN明显减少了E14、E16、E18胚胎脑Pax6阳性细胞数,但不影响Tbr2、Tbr1阳性细胞数,说明DZN的暴露主要作用于发育早期的APs,而对较为成熟的BPs和神经元影响较小。本文的结果显示,胚胎期暴露DZN能延缓APs分裂进程,扰乱APs的增殖分化平衡,减少胚胎侧脑室壁增殖池,并且这种作用能够在急性酶活力损伤作用恢复后仍然持续较长时期。更多还原

【Abstract】 Exposure to organophosphorus pesticide(OPs)diazinon(DZN)at a low concentration will affect the embryonic neural development,leading to abnormal cognitive behaviors and emotional reactions,and the specific mechanisms of DZN developmental toxicity need further study.In this research,we study the effect of DZN on apical progenitor cells(APs)from the perspective of Interkinetic nuclear migration(INM),analysis the abnormal outcome of proliferation and differentiation,and evaluate the persistence of this effect.In chapter 2,we given 0,1,5mg / kg DZN to GD14 ICR mice for three consecutive times in 3 hour intervals,measured AChE activity of maternal blood and embryos,Caspase-3 activity and SOD activity of embryos,to analysis the acute toxicity of DZN on brain tissue and the recovery.The results show that 5mg / kg DZN caused cholinesterase activity of E14,E16,E18 maternal blood decreased to 40.25%,60.34%,80.32% compared to the normal value;The cholinesterase activity of E14,E16 embryonic brain decreased to 62.53%和 79.41% to the normal value,and closed to normal value in E18;The Caspase-3 activity of E14,E16 embryonic brain increased to 148.64% and 113.86% respectively,and also closed to normal value in E18;SOD activity in three time points showed no significant change.In chapter 3,we given Brdu injection to the DZN exposed mice to mark cells in S-phase,prepared Brdu-labeled 1h,3h,6h embryos,and performed Brdu immunohistochemical staining,to study the effect of DZN on division and proliferation of Aps.The results showed that the average position of Brdu+ at 1h and 3h has no significant changes compared with control after 1mg/kg DZN exposure,and the average position of Brdu+ at 6h was slightly delyed compared to the control after 1mg/kg DZN exposure;after 5mg/kg DZN exposure,the average position of Brdu+ at 1h,3h,6h in lateral ventricle was all significantly delyed compared to the control,DZN exposure significantly slowed the INM of Brdu+.In chapter 4,the DZN exposed embryos was performed Pax6,Tbr2,Tbr1 immunohistochemical staining to analysis the effect of DZN on the development of Aps.Statistics showed that 5mg / kg DZN significantly reduced Pax6 positive cells in vertricular zone(VZ)in E14、E16、E18,but not influence the positive cell number of Tbr2 and Tbr1,indicating that DZN exposure mainly affect APs which mainly exist in the early developmental stage,but not affect BPs and neurons.Conclusively,embryonic exposure to DZN will delay the process of division of APs,disrupt the balance of proliferation and differentiation of APs,reduce the proliferating cell population,and this effect will lasts longer even thouth acute enzyme activity has recovered更多还原