本文關(guān)鍵詞：低濃度毒死蜱對小鼠胚胎神經(jīng)前體細胞周期性核運動及胚胎海馬細胞的影響　出處：《浙江工業(yè)大學》2012年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 毒死蜱 神經(jīng)前體細胞 周期性核運動 胚胎海馬

【摘要】：有機磷農(nóng)藥毒死蜱(Chlorpyrifos, CPF)能影響發(fā)育期神經(jīng)細胞增殖和分化,其作用機制尚不清楚。周期性核運動(interkinetic nuclear migration, INM)是頂端面神經(jīng)前體細胞(apical progenitors, APs)在細胞增殖周期中沿著腦室壁的頂?shù)纵S進行的核往返性移動,是胚胎神經(jīng)細胞發(fā)生中的特征性事件,也是成熟腦發(fā)育的基礎(chǔ)。海馬作為成熟腦學習記憶與行為的機能中心,形態(tài)結(jié)構(gòu)的改變與認知行為異常具有密切的關(guān)聯(lián)。本文主要從胚胎神經(jīng)前體細胞周期性核運動和胚胎期海馬形態(tài)角度評估胚胎早期CPF暴露對腦發(fā)育所產(chǎn)生的影響。 首先建立孕鼠模式,在胚胎期14天(embryo days14, E14)以3小時為間隔,連續(xù)3次皮下注射5mg/kg CPF,對照組給予等量二甲基亞砜,在首次暴露CPF0.5小時、3.5小時或5.5小時腹腔注射50mg/kg溴脫氧尿苷(5-bromo-2'-deoxyuridine, Brdu),標記S期細胞,形成不同時間段核被標記的胚胎。首次暴露CPF6.5小時處死孕鼠,剝離胚胎,多聚甲醛固定后石蠟包埋,免疫組化染色,顯微鏡下計數(shù)腦室區(qū)(ventricular Zone, VZ)和腦室下區(qū)(subvertricular zone)中Brdu標記的細胞數(shù),分析比較Brdu陽性細胞核在VZ和SVZ不同區(qū)域所占的百分比,從而評估CPF暴露對INM的即時影響。 然后在胚胎E7.5-E11.5連續(xù)5天暴露5mg/kg/d CPF, E14注射50mg/kg Brdu,分別于1,3,6,9小時處死孕鼠,胚胎腦免疫組化染色后顯微鏡下觀察,評估CPF對胚胎神經(jīng)前體細胞的INM行為是否存在滯后效應(yīng)。 為了進一步評估CPF的暴露是否通過影響海馬形態(tài)發(fā)育造成遠期認知行為學改變,實驗設(shè)計了于E7.5-E11.5暴露CPF的E16胚胎,腦組織進行冠狀位連續(xù)切片,尼氏染色,取海馬區(qū)3個標準平面,分別計數(shù)和分析細胞總數(shù)和細胞形態(tài)。 研究結(jié)果發(fā)現(xiàn),在即時暴露CPF實驗中, CPF處理使周期性核運動遷移速度變慢,具體表現(xiàn)：Brdu標記后lhr,位于VZ外層的陽性細胞核增加8.3%,而中間層減少40.1%(P0.01)；Brdu標記后3hr,位于VZ外層的陽性細胞核增加10.6%,中間層增加61.1%,而遷往VZ內(nèi)層則減少82.0%,均存在極顯著統(tǒng)計學差異(P0.01)；Brdu標記后6hr,位于VZ外層的陽性細胞核增加25.1%,中間層增加39.3%,而VZ里層減少22.2%(P0.01)。 在預先暴露CPF實驗中,CPF暴露對周期性核運動存在滯后效應(yīng),即在停止暴露CPF后,核運動速度仍然變慢。具體表現(xiàn)：Brdu標記后1hr,位于VZ外層的陽性細胞核增加6.5%,而中間層減少22.4%(P0.01);Brdu標記后3hr,位于VZ中間層增加39.8%,而遷往VZ內(nèi)層則減少25.9%,均存在極顯著統(tǒng)計學差異(P0.01)；Brdu標記后6hr,位于中間層增加15.1%,而VZ內(nèi)層減少11.7%(P0.01)。Brdu標記后9hr,位于VZ外層的陽性細胞核減少12.2%(P0.05),中間層減少22.5%(P0.01),VZ內(nèi)層無明顯改變,而SVZ區(qū)域增加95.3%,兩者之間存在極顯著差異(P0.01)。 研究胎鼠海馬形態(tài)發(fā)現(xiàn),經(jīng)過CPF處理后,海馬CAl區(qū)、CA3區(qū)細胞數(shù)量沒有明顯的變化,但比較DG區(qū)發(fā)現(xiàn),細胞數(shù)量平均減少8.06%,前者存在顯著差異,后兩者存在極顯著差異。因此對照組和CPF組DG之間細胞數(shù)量在統(tǒng)計學上存在差異。這些研究結(jié)果證明,胚胎期暴露5mg/kgCPF對胎鼠海馬形態(tài)產(chǎn)生細微的影響。
[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.

【學位授予單位】：浙江工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R384

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