Nrxn2a在铅诱导的斑马鱼神经发育毒性中的作用

发布时间：2018-08-09 07:32

【摘要】：背景:长期低剂量的铅暴露可引起神经系统损伤,对处于生长发育阶段的婴幼儿尤甚,并可进一步引起神经系统紊乱以及行为改变。在神经生长发育过程中,突触粘附分子发挥着重要的作用,但其在铅的神经发育毒性中的作用尚不明确。本研究通过观察铅暴露对斑马鱼神经系统发育的影响,以探索斑马鱼突触粘附分子在铅暴露作用下的表达改变以及在斑马鱼行为改变中的作用。方法:1.观察单细胞期不同剂量的醋酸铅(Pb(CH3COOH)2)暴露对斑马鱼胚胎/幼鱼死亡、畸形、孵化的影响;2.采用吖陡橙(Acridine Orange)染色法观察受精后3天(3 dpf)斑马鱼全鱼细胞凋亡状况;3.使用电感耦合等离子体质谱法(ICP-MS)测定铅的斑马鱼内暴露含量;4.利用ViewPoint Zebrabox行为测试系统观测斑马鱼幼鱼自发运动行为变化。5.通过q-PCR以及全鱼原位杂交(In-Situ Hybridization,ISH)技术定量及定位24 hpf,48 hpf以及72 hpf斑马鱼体内各亚型neurexin(nrxn)以及72hpf部分突触粘附分子mRNA表达水平与空间表达状况;6.采用显微注射技术于斑马鱼胚胎单细胞期注射人工合成的nrxn2abmRNA进行进行拯救实验(rescue experiment),进一步观察过表达nrxn2abmRNA后,铅暴露下斑马鱼胚胎/幼鱼的死亡、畸形、孵化状况,凋亡发生,nrxn2aa与nrxn2ab表达变化以及行为学改变。结果:1.随着铅暴露剂量的增加,斑马鱼胚胎死亡率和畸形率有不同程度的升高。持续暴露至120hpf时,最高受试剂量(10μmol/L)引起斑马鱼幼鱼死亡率约为18%;铅暴露可使斑马鱼出现多种畸形表型,主要表现为脊柱弯曲畸形,于120hpf畸形率达26%。2.AO染色凋亡显示斑马鱼幼鱼凋亡水平随着暴露剂量的增加而升高,其中凋亡发生主要聚集于头部、心包以及鱼鳍等区域。在NBT-DsRed转基因鱼系(特异性标记神经元)中,铅暴露引起较明显的神经系统凋亡;3.ICP-MS结果显示铅有较强的生物蓄积作用,并伴有剂量效应关系;4.在行为学观察中,可见在受试浓度下的斑马鱼的自发运动受到了抑制,表现为总运动位移的减少、活动比例以及平均速度的降低,并随着暴露剂量的升高而运动行为抑制增加。5.通过qPCR检测铅暴露对受试突触粘附分子mRNA水平表达的影响,结果显示仅nrxn2a基因表达受到铅暴露的影响。qPCR和原位杂交实验展示了斑马鱼各亚型的nrxn的mRNA水平表达状况。铅暴露于各测试时间点对nrxn2aa和nrxn2ab的表达均具有较明显的抑制作用。其中铅暴露于24 hpf诱导了nrxnla和nrxn3a的表达并于48 hpf产生抑制作用,而至72hpf则无明显改变。6.在拯救实验中,我们观察nrxn2abmRNA注射后斑马鱼胚胎生长发育状况,发现mRNA注射可明显减少铅暴露导致的脊柱畸形发生同时降低神经系统(NBT-dsRed转基因鱼)和全鱼(野生型)凋亡水平。在行为学观察中,mRNA的过表达可恢复斑马鱼幼鱼的运动水平。qPCR检测结果显示nrxn2abmRNA的过表达同样上调了nrxn2a 的mRNA表达。结论:铅暴露可以诱导斑马鱼nrxn2a基因表达下调,同时引起自发运动行为学改变。铅在不同暴露时间点稳定下调nrxn2a基因,并诱导神经系统凋亡发生。结合rescue结果,本研究确认了nrxn2 在斑马鱼神经系统中的重要作用,并能够进一步影响斑马鱼自发运动行为。
[Abstract]:Background: long term low dose lead exposure can cause nervous system damage, especially for infants at the growth stage, which may further cause nervous system disorders and behavioral changes. In the process of nerve growth, synaptic adhesion molecules play an important role, but their role in the neurodevelopmental toxicity of lead is not clear. In this study, the effects of lead exposure on the development of zebrafish nervous system were observed to explore the expression changes of zebrafish synaptic adhesion molecules under lead exposure and the role of zebrafish behavior. Methods: 1. the death, deformity and incubation of different doses of lead acetate (Pb (CH3COOH) 2) on zebrafish embryos / young fish were observed. 2. Acridine Orange staining was used to observe the apoptosis of all fish cells in zebrafish 3 days (3 DPF) after fertilization and 3. using inductively coupled plasma mass spectrometry (ICP-MS) to determine the exposure content of zebrafish in lead, and 4. using the ViewPoint Zebrabox test system to observe the spontaneous movement behavior of zebrafish fish.5. Q-PCR and total fish in situ hybridization (In-Situ Hybridization, ISH) techniques were used to quantify and locate the mRNA expression level and space expression level of the synapses in each subtype neurexin (NRXN) and 72hpf part of the 24 hpf, 48 HPF and 72 HPF zebrafish. 6. microinjection technique was injected into the single cell stage of zebrafish embryos by injecting the synthetic nrxn2a BmRNA carried out the rescue experiment (rescue experiment) to further observe the death, deformity, hatching status, apoptosis, nrxn2aa and nrxn2ab expression changes and behavioral changes of zebrafish embryos / young fish exposed to lead exposure. Results: 1. as the dosage of lead exposure increased, the mortality and malformation rate of zebrafish embryos were not At the same level, the maximum trial dose (10 u mol/L) caused the mortality of zebrafish young fish about 18%, and lead exposure could cause a variety of deformity phenotypes of zebrafish, mainly manifested as spinal curvature, and 26%.2.AO staining apoptosis at 120hpf malformation rate showed that the apoptosis level of zebrafish fish increased with the increase of exposure dose. The apoptosis occurred mainly in the head, the pericardium and the fish fins. In the NBT-DsRed transgenic fish system (specific labeled neurons), the lead exposure caused the obvious neuronal apoptosis; the results of 3.ICP-MS showed that lead had stronger biological accumulation and had a dose effect relationship. 4. in behavioral observation, it was seen in the subject of behavioral observation. The spontaneous movement of zebrafish under test concentration was inhibited, showing the decrease of total movement displacement, activity ratio and average velocity, and the effect of.5. on the horizontal expression of mRNA in the tested synapse by qPCR detection with the increase of exposure dose. The result showed that only nrxn2a gene table was found. The effects of lead exposure on.QPCR and in situ hybridization showed the mRNA level expression of NRXN in each subtype of zebrafish. Lead exposure was significantly inhibited on the expression of nrxn2aa and nrxn2ab at each test time point. Lead exposure to 24 hpf induced the expression of nrxnla and nrxn3a and produced a inhibitory effect on 48 HPF, To 72hpf, there was no significant change in.6. in the rescue experiment. We observed the growth and development of zebrafish embryos after nrxn2abmRNA injection. It was found that mRNA injection could significantly reduce the incidence of spinal deformities caused by lead exposure and reduce the apoptosis level of the nervous system (NBT-dsRed transgenic fish) and all fish (wild type). In behavioral observation, the overexpression of mRNA The results of.QPCR detection of the recoverable zebrafish young fish showed that the overexpression of nrxn2abmRNA also increased the mRNA expression of nrxn2a. Conclusion: lead exposure can induce the down regulation of nrxn2a gene expression in zebrafish, and cause spontaneous locomotor behavior changes. Lead at different exposure points can stabilize the nrxn2a gene and induce the nervous system withering. In combination with the results of rescue, this study confirmed the important role of nrxn2 in the nervous system of zebrafish and further affected the spontaneous movement behavior of zebrafish.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R114

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