低浓度毒死蜱对小鼠胚胎神经前体细胞周期性核运动及胚胎海马细胞的影响

发布时间：2018-01-05 15:16

本文关键词：低浓度毒死蜱对小鼠胚胎神经前体细胞周期性核运动及胚胎海马细胞的影响　出处：《浙江工业大学》2012年硕士论文　论文类型：学位论文

【摘要】：有机磷农药毒死蜱(Chlorpyrifos, CPF)能影响发育期神经细胞增殖和分化,其作用机制尚不清楚。周期性核运动(interkinetic nuclear migration, INM)是顶端面神经前体细胞(apical progenitors, APs)在细胞增殖周期中沿着脑室壁的顶底轴进行的核往返性移动,是胚胎神经细胞发生中的特征性事件,也是成熟脑发育的基础。海马作为成熟脑学习记忆与行为的机能中心,形态结构的改变与认知行为异常具有密切的关联。本文主要从胚胎神经前体细胞周期性核运动和胚胎期海马形态角度评估胚胎早期CPF暴露对脑发育所产生的影响。首先建立孕鼠模式,在胚胎期14天(embryo days14, E14)以3小时为间隔,连续3次皮下注射5mg/kg CPF,对照组给予等量二甲基亚砜,在首次暴露CPF0.5小时、3.5小时或5.5小时腹腔注射50mg/kg溴脱氧尿苷(5-bromo-2'-deoxyuridine, Brdu),标记S期细胞,形成不同时间段核被标记的胚胎。首次暴露CPF6.5小时处死孕鼠,剥离胚胎,多聚甲醛固定后石蜡包埋,免疫组化染色,显微镜下计数脑室区(ventricular Zone, VZ)和脑室下区(subvertricular zone)中Brdu标记的细胞数,分析比较Brdu阳性细胞核在VZ和SVZ不同区域所占的百分比,从而评估CPF暴露对INM的即时影响。然后在胚胎E7.5-E11.5连续5天暴露5mg/kg/d CPF, E14注射50mg/kg Brdu,分别于1,3,6,9小时处死孕鼠,胚胎脑免疫组化染色后显微镜下观察,评估CPF对胚胎神经前体细胞的INM行为是否存在滞后效应。为了进一步评估CPF的暴露是否通过影响海马形态发育造成远期认知行为学改变,实验设计了于E7.5-E11.5暴露CPF的E16胚胎,脑组织进行冠状位连续切片,尼氏染色,取海马区3个标准平面,分别计数和分析细胞总数和细胞形态。研究结果发现,在即时暴露CPF实验中, CPF处理使周期性核运动迁移速度变慢,具体表现：Brdu标记后lhr,位于VZ外层的阳性细胞核增加8.3%,而中间层减少40.1%(P0.01)；Brdu标记后3hr,位于VZ外层的阳性细胞核增加10.6%,中间层增加61.1%,而迁往VZ内层则减少82.0%,均存在极显著统计学差异(P0.01)；Brdu标记后6hr,位于VZ外层的阳性细胞核增加25.1%,中间层增加39.3%,而VZ里层减少22.2%(P0.01)。在预先暴露CPF实验中,CPF暴露对周期性核运动存在滞后效应,即在停止暴露CPF后,核运动速度仍然变慢。具体表现：Brdu标记后1hr,位于VZ外层的阳性细胞核增加6.5%,而中间层减少22.4%(P0.01);Brdu标记后3hr,位于VZ中间层增加39.8%,而迁往VZ内层则减少25.9%,均存在极显著统计学差异(P0.01)；Brdu标记后6hr,位于中间层增加15.1%,而VZ内层减少11.7%(P0.01)。Brdu标记后9hr,位于VZ外层的阳性细胞核减少12.2%(P0.05),中间层减少22.5%(P0.01),VZ内层无明显改变,而SVZ区域增加95.3%,两者之间存在极显著差异(P0.01)。研究胎鼠海马形态发现,经过CPF处理后,海马CAl区、CA3区细胞数量没有明显的变化,但比较DG区发现,细胞数量平均减少8.06%,前者存在显著差异,后两者存在极显著差异。因此对照组和CPF组DG之间细胞数量在统计学上存在差异。这些研究结果证明,胚胎期暴露5mg/kgCPF对胎鼠海马形态产生细微的影响。
[Abstract]:Organophosphorus pesticide chlorpyrifos (Chlorpyrifos, CPF) can affect the development of cell proliferation and differentiation, its mechanism is still unclear. The periodic nuclear movement (interkinetic nuclear migration, INM) is the top surface of neural precursor cells (apical, progenitors, APs) on the cell proliferation cycle along the axis of the top and bottom wall of ventricle the return of nuclear movement, is a feature of the event occurred in the embryonic nerve cells, and also is the foundation of mature brain development. The hippocampus as mature brain learning and memory behavior function of the center, closely associated with abnormal morphological changes and cognitive behavior. This article mainly from the embryonic neural precursor cells and embryonic nuclear periodic motion assessment of hippocampal morphology CPF exposure early embryo effects on brain development are produced.
The first model of pregnant rats at embryonic day 14 (embryo, days14, E14) at intervals of 3 hours, 3 consecutive subcutaneous injection of 5mg/kg CPF, the control group received the same amount of two dimethyl sulfoxide, at first exposure CPF0.5 hours, 3.5 hours or 5.5 hours 50mg/kg intraperitoneal injection of bromodeoxyuridine (5-bromo-2'-deoxyuridine, Brdu), mark the cells in S phase, the formation of different time nuclear labeled embryos. The first exposure of pregnant rats were killed at CPF6.5 hours, stripping embryos, fixed in paraformaldehyde and embedded in paraffin. Immunohistochemical staining, counting the ventricular zone under the microscope (ventricular Zone, VZ) and the subventricular zone (subvertricular zone) the number of cells labeled with Brdu the percentage of Brdu positive nuclei, comparative analysis for different regions of VZ and SVZ, to assess the exposure of CPF instant effect on INM.
Then embryo E7.5-E11.5 was exposed to 5mg/kg/d CPF for 5 consecutive days, E14 was injected into 50mg/kg Brdu, and the pregnant rats were killed at 1,3,6,9 hour. After embryonic brain immunohistochemical staining, we observed the delayed effect of CPF on INM behavior of embryonic neural progenitor cells under microscope.
In order to further evaluate the impact of CPF exposure through long-term cognitive behavior changes of hippocampus development form, experimental design in E7.5-E11.5 exposed CPF E16 embryonic brain tissue, coronal serial sections and Nissl staining, 3 standard plane hippocampus, were counted and analyzed the total number of cells and cell morphology.
The results of the study showed that in the instant exposure in the CPF experiment, CPF treatment make periodic nuclear migration slows down, the specific performance: Brdu labeled LHR positive nuclei in VZ layer increased by 8.3%, while the middle layer is reduced by 40.1% (P0.01); Brdu marker 3hr, located in the outer layer of the VZ positive cells increased 10.6%, intermediate the 61.1% layer increases, and moved to the inner layer of VZ is reduced by 82%, there were significant statistical difference (P0.01); Brdu marker 6hr, located in the outer layer of the VZ positive cells increased by 25.1%, the middle layer and the VZ layer increased by 39.3%, reduced by 22.2% (P0.01).
In the pre exposure CPF experiment, CPF exposure has lag effect of periodic nuclear movement, namely in the stop after exposure to CPF, nuclear movement speed is still slow. Specific performance: Brdu labeled 1hr positive nuclei in VZ layer increased by 6.5%, while the middle layer is reduced by 22.4% (P0.01); Brdu marker 3hr, located in the the middle layer of VZ increased by 39.8%, and moved to the inner layer of VZ is reduced by 25.9%, there were significant statistical difference (P0.01); Brdu marker 6hr, located in the middle layer and the inner layer is increased by 15.1%, VZ decreased by 11.7% (P0.01).Brdu labeled 9hr positive nuclei in VZ layer decreased by 12.2% (P0.05), the middle layer is reduced by 22.5% (P0.01 VZ), but no significant changes in the inner, and the SVZ region increased 95.3%, there was significant difference between them (P0.01).
Study on hippocampal morphology of fetal rats found that after treatment with CPF in hippocampal CAl region, the number of CA3 cells did not change obviously, but the DG found in the area, the number of cells was reduced by 8.06% in average, there are significant differences in the former, the latter two had extremely significant difference. So the number of cells between the control group and CPF group DG existed in statistics difference. These results demonstrated that embryonic exposure to 5mg/kgCPF have subtle effects on hippocampal morphology of fetal rats.

【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R384

【共引文献】