Nrxn2a在鉛誘導(dǎo)的斑馬魚神經(jīng)發(fā)育毒性中的作用
發(fā)布時(shí)間：2018-08-09 07:32
【摘要】：背景:長(zhǎng)期低劑量的鉛暴露可引起神經(jīng)系統(tǒng)損傷,對(duì)處于生長(zhǎng)發(fā)育階段的嬰幼兒尤甚,并可進(jìn)一步引起神經(jīng)系統(tǒng)紊亂以及行為改變。在神經(jīng)生長(zhǎng)發(fā)育過(guò)程中,突觸粘附分子發(fā)揮著重要的作用,但其在鉛的神經(jīng)發(fā)育毒性中的作用尚不明確。本研究通過(guò)觀察鉛暴露對(duì)斑馬魚神經(jīng)系統(tǒng)發(fā)育的影響,以探索斑馬魚突觸粘附分子在鉛暴露作用下的表達(dá)改變以及在斑馬魚行為改變中的作用。方法:1.觀察單細(xì)胞期不同劑量的醋酸鉛(Pb(CH3COOH)2)暴露對(duì)斑馬魚胚胎/幼魚死亡、畸形、孵化的影響;2.采用吖陡橙(Acridine Orange)染色法觀察受精后3天(3 dpf)斑馬魚全魚細(xì)胞凋亡狀況;3.使用電感耦合等離子體質(zhì)譜法(ICP-MS)測(cè)定鉛的斑馬魚內(nèi)暴露含量;4.利用ViewPoint Zebrabox行為測(cè)試系統(tǒng)觀測(cè)斑馬魚幼魚自發(fā)運(yùn)動(dòng)行為變化。5.通過(guò)q-PCR以及全魚原位雜交(In-Situ Hybridization,ISH)技術(shù)定量及定位24 hpf,48 hpf以及72 hpf斑馬魚體內(nèi)各亞型neurexin(nrxn)以及72hpf部分突觸粘附分子mRNA表達(dá)水平與空間表達(dá)狀況;6.采用顯微注射技術(shù)于斑馬魚胚胎單細(xì)胞期注射人工合成的nrxn2abmRNA進(jìn)行進(jìn)行拯救實(shí)驗(yàn)(rescue experiment),進(jìn)一步觀察過(guò)表達(dá)nrxn2abmRNA后,鉛暴露下斑馬魚胚胎/幼魚的死亡、畸形、孵化狀況,凋亡發(fā)生,nrxn2aa與nrxn2ab表達(dá)變化以及行為學(xué)改變。結(jié)果:1.隨著鉛暴露劑量的增加,斑馬魚胚胎死亡率和畸形率有不同程度的升高。持續(xù)暴露至120hpf時(shí),最高受試劑量(10μmol/L)引起斑馬魚幼魚死亡率約為18%;鉛暴露可使斑馬魚出現(xiàn)多種畸形表型,主要表現(xiàn)為脊柱彎曲畸形,于120hpf畸形率達(dá)26%。2.AO染色凋亡顯示斑馬魚幼魚凋亡水平隨著暴露劑量的增加而升高,其中凋亡發(fā)生主要聚集于頭部、心包以及魚鰭等區(qū)域。在NBT-DsRed轉(zhuǎn)基因魚系(特異性標(biāo)記神經(jīng)元)中,鉛暴露引起較明顯的神經(jīng)系統(tǒng)凋亡;3.ICP-MS結(jié)果顯示鉛有較強(qiáng)的生物蓄積作用,并伴有劑量效應(yīng)關(guān)系;4.在行為學(xué)觀察中,可見在受試濃度下的斑馬魚的自發(fā)運(yùn)動(dòng)受到了抑制,表現(xiàn)為總運(yùn)動(dòng)位移的減少、活動(dòng)比例以及平均速度的降低,并隨著暴露劑量的升高而運(yùn)動(dòng)行為抑制增加。5.通過(guò)qPCR檢測(cè)鉛暴露對(duì)受試突觸粘附分子mRNA水平表達(dá)的影響,結(jié)果顯示僅nrxn2a基因表達(dá)受到鉛暴露的影響。qPCR和原位雜交實(shí)驗(yàn)展示了斑馬魚各亞型的nrxn的mRNA水平表達(dá)狀況。鉛暴露于各測(cè)試時(shí)間點(diǎn)對(duì)nrxn2aa和nrxn2ab的表達(dá)均具有較明顯的抑制作用。其中鉛暴露于24 hpf誘導(dǎo)了nrxnla和nrxn3a的表達(dá)并于48 hpf產(chǎn)生抑制作用,而至72hpf則無(wú)明顯改變。6.在拯救實(shí)驗(yàn)中,我們觀察nrxn2abmRNA注射后斑馬魚胚胎生長(zhǎng)發(fā)育狀況,發(fā)現(xiàn)mRNA注射可明顯減少鉛暴露導(dǎo)致的脊柱畸形發(fā)生同時(shí)降低神經(jīng)系統(tǒng)(NBT-dsRed轉(zhuǎn)基因魚)和全魚(野生型)凋亡水平。在行為學(xué)觀察中,mRNA的過(guò)表達(dá)可恢復(fù)斑馬魚幼魚的運(yùn)動(dòng)水平。qPCR檢測(cè)結(jié)果顯示nrxn2abmRNA的過(guò)表達(dá)同樣上調(diào)了nrxn2a 的mRNA表達(dá)。結(jié)論:鉛暴露可以誘導(dǎo)斑馬魚nrxn2a基因表達(dá)下調(diào),同時(shí)引起自發(fā)運(yùn)動(dòng)行為學(xué)改變。鉛在不同暴露時(shí)間點(diǎn)穩(wěn)定下調(diào)nrxn2a基因,并誘導(dǎo)神經(jīng)系統(tǒng)凋亡發(fā)生。結(jié)合rescue結(jié)果,本研究確認(rèn)了nrxn2 在斑馬魚神經(jīng)系統(tǒng)中的重要作用,并能夠進(jìn)一步影響斑馬魚自發(fā)運(yùn)動(dòng)行為。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114
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