本文選題：砷 + 小腦�。� 參考：《大連醫(yī)科大學(xué)》2014年博士論文

【摘要】：背景及目的砷(Arsenic, As)是一種類金屬,以無機(jī)砷和有機(jī)砷兩種形態(tài)在自然界廣泛分布。慢性砷中毒已成為影響數(shù)百萬人健康的世界性公共衛(wèi)生問題。在砷污染地區(qū),飲用水或地下水中砷濃度達(dá)到0.25-2.1 ppm,污染嚴(yán)重的地方砷濃度已超過3.0ppm。孟加拉國、印度和中國是全世界受危害人口最多、病區(qū)面積最大、病情最嚴(yán)重的國家。我國飲水型砷中毒病區(qū)高砷暴露人群接近300萬,且不包括由于燃煤引起的燃煤型地方性砷中毒。砷暴露對(duì)健康的危害是多方面的,慢性砷中毒可致引發(fā)多器官的組織學(xué)與功能上的異常改變,包括呼吸道、消化道、肝臟、腎臟、皮膚組織、免疫系統(tǒng)以及神經(jīng)系統(tǒng)。神經(jīng)細(xì)胞具有不能再生的特點(diǎn),與其他組織比較神經(jīng)細(xì)胞對(duì)毒物的毒性作用更為敏感,因此砷暴露對(duì)神經(jīng)系統(tǒng)的毒性倍受關(guān)注。一些研究報(bào)道,急慢性砷暴露兒童會(huì)出現(xiàn)與神經(jīng)系統(tǒng)損傷有關(guān)后遺癥。由于腦功能受到抑制,從而導(dǎo)致神經(jīng)組織發(fā)育障礙和智力低下,例如出現(xiàn)運(yùn)動(dòng)、認(rèn)知、心理、感覺和語言等功能障礙。對(duì)孟加拉國砷含量偏高地區(qū)進(jìn)行的兩項(xiàng)流行病學(xué)調(diào)查顯示,兒童水砷暴露與智力降低密切相關(guān),與染砷動(dòng)物實(shí)驗(yàn)結(jié)果一致。提示,砷中毒影響青少年學(xué)習(xí)記憶能力。然而,砷暴露影響學(xué)習(xí)記憶能力的機(jī)理目前尚不十分清楚。動(dòng)物試驗(yàn)結(jié)果表明,砷暴露可導(dǎo)致小鼠小腦中砷含量呈劑量依賴性增高,且損傷小腦神經(jīng)元,影響小腦功能。這些結(jié)果提示,小腦可能是砷毒性作用的靶器官之一。研究顯示：小腦不僅負(fù)責(zé)調(diào)解肌張力、保持身體平衡和協(xié)調(diào)精細(xì)運(yùn)動(dòng),而且參與認(rèn)知功能的調(diào)節(jié),包括學(xué)習(xí)能力、注意集中力、語言能力等。上述動(dòng)物試驗(yàn)結(jié)果和文獻(xiàn)報(bào)道表明,損傷性影響小腦功能可能與砷暴露導(dǎo)致的學(xué)習(xí)記憶能力下降有關(guān)。有研究報(bào)道鉛、鋁、氟等暴露可導(dǎo)致學(xué)習(xí)記憶能力下降。而且已證實(shí)鉛、鋁、氟的神經(jīng)毒性與調(diào)控學(xué)習(xí)記憶機(jī)制相關(guān)蛋白的編碼基因表達(dá)變化有關(guān)。近年來,越來越多的學(xué)者開始把小腦作為神經(jīng)毒物靶器官開展研究。那么,砷的神經(jīng)毒作用機(jī)制是否與小腦學(xué)習(xí)記憶相關(guān)的基因表達(dá)紊亂有關(guān)呢,引起我們極大興趣�；蛐酒夹g(shù)因具有快速、準(zhǔn)確、同時(shí)分析大量基因信息的特點(diǎn),被廣泛運(yùn)用于生命科學(xué)領(lǐng)域。采用基因芯片技術(shù)研究在環(huán)境毒物刺激條件下的大量基因差異表達(dá)情況,可揭示神經(jīng)毒性相關(guān)的分子生物機(jī)制。運(yùn)用基因芯片技術(shù)分析砷暴露所致小腦基因表達(dá)譜變化,對(duì)篩選砷所致神經(jīng)毒性相關(guān)基因及其探討可能機(jī)制有著重要的作用。本研究采用成年小鼠進(jìn)行飲水砷染毒,并觀察亞慢性砷暴露對(duì)小鼠小腦學(xué)習(xí)記憶能力的影響；采用基因芯片技術(shù)篩選出砷暴露組與對(duì)照組小鼠小腦的差異表達(dá)基因,經(jīng)通路分析發(fā)現(xiàn)長時(shí)程記憶等相關(guān)通路受到影響；在篩選出的差異表達(dá)基因中,發(fā)現(xiàn)突觸后致密體功能基因表達(dá)受到影響,包括離子型／膜型谷氨酸受體、IP3受體、激酶CaMKⅡ、PLCβ4等；由于這些基因在長時(shí)程記憶相關(guān)通路中起到關(guān)鍵的作用,引起我們極大興趣,我們觀察了小腦突觸后致密體超微結(jié)構(gòu),且對(duì)這些基因的表達(dá)進(jìn)行驗(yàn)證。并檢測突觸后致密體關(guān)鍵酶CaMKⅡ的蛋白表達(dá)水平,且對(duì)CaMKⅡ下游長時(shí)程記憶相關(guān)通路的關(guān)鍵蛋白進(jìn)行檢測。為闡明砷致小腦學(xué)習(xí)記憶功能損害的分子機(jī)制以及防治砷中毒提供依據(jù)。方法1、選用月齡為一個(gè)月的健康昆明小鼠64只,體重22.3-26.4 g,在標(biāo)準(zhǔn)動(dòng)物房內(nèi)飼養(yǎng)。對(duì)照組小鼠飲用滅菌自來水,實(shí)驗(yàn)組小鼠分別飲用1 ppm、2 ppm和4 ppm三氧化二砷水溶液。小鼠飲用水每兩天更換一次,染毒時(shí)間為2個(gè)月。2、測定腦砷含量：采用電感耦合等離子體質(zhì)譜(ICP-MS)測量方法。3、HE染色觀察小腦組織形態(tài)學(xué)變化。4、檢測小鼠學(xué)習(xí)記憶能力和小腦運(yùn)動(dòng)性學(xué)習(xí)記憶能力：分別采用Morris水迷宮定向航行試驗(yàn)和瞬膜條件反射試驗(yàn)。5、小鼠小腦組織基因芯片檢測,差異表達(dá)基因分析。6、檢測亞慢性砷暴露對(duì)小鼠小腦突觸后致密體基因表達(dá)影響。采用real time-PCR(RT-PCR)檢測小腦NMDA受體亞基NR1、AMPA受體亞基GluR1和GluR2、mGlu受體亞基mGluR1、IP3受體亞基IP3R1、激酶CaMKⅡ、PLCβ4基因表達(dá)。7、透射電鏡觀察小鼠小腦組織突觸形態(tài)結(jié)構(gòu)的變化。8、檢測小腦CaMKⅡ及下游通路關(guān)鍵蛋白表達(dá)變化。采用免疫印跡法(Western-blot)檢測小鼠小腦PSD中CaMKⅡ蛋白表達(dá)和磷酸化水平；檢測小鼠小腦組織Ras、Raf、ERK1/2的磷酸化水平。結(jié)果1、實(shí)驗(yàn)期間,各組小鼠一般情況良好。隨飼養(yǎng)時(shí)間延長,小鼠體重增加。從第36天開始,4 ppm劑量組小鼠體重增幅減慢,與對(duì)照組和1 ppm染砷組相比,差異有統(tǒng)計(jì)學(xué)(P0.05)。這種差異一直維持到實(shí)驗(yàn)結(jié)束。在染毒末期,2 ppm劑量組小鼠體重增幅減慢,與對(duì)照組和1 ppm染砷組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2、各實(shí)驗(yàn)組小鼠小腦砷含量均顯著高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)且呈劑量依賴關(guān)系。3、本研究高劑量染砷組中,小腦分子細(xì)胞層胞漿染色不均勻,纖維分布不均,細(xì)胞漿淡染,細(xì)胞皺縮。浦肯野細(xì)胞層,小腦組織浦肯野細(xì)胞層胞漿染色淡,纖維(粉染區(qū))變大,且不均勻,細(xì)胞胞漿淡染,胞核皺縮；顆粒細(xì)胞層細(xì)胞數(shù)量減少。4、亞慢性砷暴露對(duì)小鼠運(yùn)動(dòng)性學(xué)習(xí)記憶能力的影響Morris水迷宮結(jié)果：對(duì)小鼠采用As203染毒60天后,與對(duì)照組比較：2 ppm和4 ppm As2O3染毒組小鼠的逃避潛伏期顯著增長(P0.05),并且逃避潛伏期隨著染毒劑量的增加而增長。瞬膜條件反射結(jié)果提示：As203染毒60天后,4 ppm As2O3染毒小鼠的瞬膜條件反射與對(duì)照組比較呈顯著降低(P0.05)。5、基因芯片結(jié)果顯示,與對(duì)照組小鼠小腦基因表達(dá)譜比較,4 ppm染砷組顯示1597個(gè)差異表達(dá)基因,其中1046個(gè)表達(dá)下調(diào),551個(gè)表達(dá)上調(diào)。GO分析提示,突觸后致密體上蛋白編碼基因的表達(dá)有顯著性差異,KEGG通路分析LTP和LTD為有顯著性差異(且P值最小)的通路。位于突觸后致密體并參與LTP/LTD的基因?yàn)椋篊aMKⅡ、Gria1、Gria2、Grin1、Itpr1、Grm1、PLCβ4基因。6、與基因芯片結(jié)果一致,CaMKⅡ、Gria1、Gria2、Grin1、Itpr1、Grm1、PLCβ4的mRNA表達(dá)下降。7、透射電鏡觀察發(fā)現(xiàn),As203染毒60天后,2 ppm和4 ppm As2O3染毒小鼠的突觸后致密體厚度與對(duì)照組比較顯著變薄(P0.05)。8、亞慢性砷暴露后,小鼠小腦CaMKⅡ 蛋白表達(dá)及磷酸化狀態(tài)蛋白表達(dá)都下降(P0.05),磷酸化Raf, ERK1/2蛋白表達(dá)下降(P0.05)。結(jié)論1、亞慢性砷暴露后,砷在小鼠腦組織中蓄積,呈劑量反應(yīng)關(guān)系。2、亞慢性砷暴露導(dǎo)致小腦組織形態(tài)學(xué)異常改變,亞慢性砷暴露損害學(xué)習(xí)記憶功能。3、亞慢性砷暴露干擾小腦學(xué)習(xí)記憶功能相關(guān)基因表達(dá)。4、亞慢性砷暴露使小鼠小腦PSD功能基因NR1、Gria1、Gria2、mGluR1、 Itpr1、PLCβ4、CaMKⅡ表達(dá)顯著異常；導(dǎo)致小腦組織PSD厚度改變。5、亞慢性砷暴露抑制小鼠小腦CaMKⅡ及其下游信號(hào)通路活性可能是砷神經(jīng)毒作用的一個(gè)重要機(jī)制。
[Abstract]:Background and objective Arsenic (As) is a kind of metal, which is widely distributed in nature with two forms of inorganic and organic arsenic. Chronic arsenism has become a worldwide public health problem affecting the health of millions of people. Arsenic concentration in drinking water or groundwater reaches 0.25-2.1 ppm in arsenic polluted areas, and the concentration of arsenic in serious polluted places has been exceeded. 3.0ppm. Bangladesh, India and China are the most endangered species in the world, the area with the largest disease area and the most serious condition. The high arsenic exposure population in the drinking water type arsenism area of our country is close to 3 million, and it does not include coal burning endemic arsenism caused by coal burning. Abnormal changes in histology and function that cause multiple organs, including respiratory tract, digestive tract, liver, kidney, skin tissue, immune system, and nervous system. Nerve cells are not regenerative and are more sensitive to the toxicity of nerve cells to other tissues, so the toxicity of arsenic exposure to the nervous system is greatly affected. Attention. Some studies have reported that children with acute and chronic arsenic exposure may have sequelae associated with nervous system injuries. The inhibition of brain function leads to neurological dysplasia and mental retardation, such as motor, cognitive, psychological, sensory, and language dysfunction. The two epidemic of high arsenic content in Bangladesh The disease survey showed that arsenic exposure in children was closely related to the decrease of intelligence, which was consistent with the experimental results of arsenic contamination. It was suggested that arsenic poisoning affects the learning and memory ability of adolescents. However, the mechanism of arsenic exposure affects learning and memory ability is not very clear. Animal test results show that arsenic exposure can lead to arsenic content in mice in the cerebellum. The results suggest that the cerebellum is not only responsible for mediating muscle tension, maintaining balance and coordinating fine movement, but also regulating cognitive function, including learning, concentration, and language ability. The results of these animal tests and literature have shown that the damage to the cerebellar function may be related to the decline in learning and memory ability caused by arsenic exposure. Studies have reported that exposure to lead, aluminum, fluoride and other exposure can lead to a decline in learning and memory. In recent years, more and more scholars have begun to study the cerebellum as the target organ of nerve poison. Then, whether the mechanism of arsenic neurotoxicity is related to the disorder of gene expression related to learning and memory in the cerebellum, which has aroused great interest. The characteristics of it are widely used in the field of life science. Using gene chip technology to study a large number of gene differential expressions under the environment of environmental toxicants, we can reveal the molecular biological mechanism related to neurotoxicity. Using gene chip technology to analyze the changes in the gene spectrum of cerebellum caused by arsenic exposure and to screen the neurotoxicity related to arsenic. The gene and its possible mechanism have an important role. In this study, the effects of arsenic exposure on drinking water in adult mice and the effect of subchronic arsenic exposure on the learning and memory ability of cerebellum in mice were observed. Gene chip technology was used to screen the differentially expressed genes in the cerebellum of the arsenic exposure group and the control group, and the long history was found through the pathway analysis. Memory and other related pathways were affected; in the screened differentially expressed genes, the expression of the functional gene of the postsynaptic dense body was found to be affected, including the ionic / membranous glutamic acid receptor, IP3 receptor, kinase CaMK II, PLC beta 4, and so on, because these genes play a key role in the long term memory related pathways, which are of great interest to us, We observed the ultrastructure of the postsynaptic dense body of the cerebellum and verified the expression of these genes, and detected the protein expression level of the key enzyme CaMK II of the postsynaptic densification, and the detection of the key proteins in the long term memory related pathway of the downstream CaMK II, to clarify the molecular mechanism of the impairment of learning and memory function of arsenic induced small brain. Methods 1, methods 1, 64 healthy Kunming mice with a month old age were selected and the mice were fed in the standard animal room with a weight of 22.3-26.4 G. The mice in the control group drank 1 ppm, 2 ppm and 4 ppm arsenic trioxide in the experimental mice. The mice were replaced every two days and the time of poisoning was 2. Month.2, determination of brain arsenic content: using inductively coupled plasma mass spectrometry (ICP-MS) measurement method.3, HE staining to observe the morphological changes of cerebellum.4, test the learning and memory ability of mice and the ability of cerebellar motor learning and memory: Morris water maze directional navigation test and instant membrane conditioned reflex test.5, mouse cerebellar tissue gene, respectively. Microarray detection and differential expression gene analysis.6 were used to detect the effect of sub chronic arsenic exposure on the gene expression of postsynaptic dense body in cerebellum of mice. Real time-PCR (RT-PCR) was used to detect the cerebellar NMDA receptor subunit NR1, AMPA receptor subunit GluR1 and GluR2, mGlu receptor subunit mGluR1, IP3 receptor subunit, kinase 4 gene expression and transmission electron microscopy The changes in the morphological structure of synapses in the cerebellar tissues of mice were detected by.8, and the changes in the expression of key proteins in the cerebellar CaMK II and downstream pathways were detected. The expression and phosphorylation of CaMK II protein in the cerebellar PSD of mice were detected by immunoblotting (Western-blot), and the phosphorylation level of Ras, Raf, ERK1 /2 in the cerebellar tissues of mice was detected. Results 1, during the experimental period, mice in each group The general situation was good. The weight gain of mice increased with the feeding time. From thirty-sixth days, the weight gain of mice in the 4 ppm dose group slowed down, compared with the control group and the 1 ppm arsenic group, the difference was statistically significant (P0.05). The difference of weight in the 2 ppm dose group slowed down with the control group and 1 ppm at the end of the poisoning. Compared with the arsenic group, the difference was statistically significant (P0.05).2, and the arsenic content in cerebellum of all the experimental groups was significantly higher than that of the control group. The difference was statistically significant (P0.05) and was dose dependent.3. In the high dose arsenic staining group, the cytoplasmic staining of the cerebellar molecular cell layer was uneven, the fiber distribution was uneven, the cytoplasm light dye, the cell shrinkage. Purkinje fine. In the cell layer, the cytoplasm of the Purkinje cell layer of the cerebellum was dyed, the fiber (powder dyeing area) became larger and uneven, the cell cytoplasm was pale, the nucleus crinkled, the number of cell layer cells decreased by.4, and the effect of subchronic arsenic exposure on the ability of motor learning and memory in mice was Morris water maze: 60 days after 60 days of exposure to the mice, the mice were compared with the control group. The escape latency of 2 ppm and 4 ppm As2O3 mice increased significantly (P0.05), and the escape latency increased with the increase of the dose. The result of the instant membrane conditioned reflex suggested that the instant membrane condition reflex of 4 ppm As2O3 infected mice was significantly lower than the control group (P0.05).5, and the gene chip results showed a significant difference for 60 days. Compared with the cerebellar gene expression profiles in the control group, 4 ppm arsenic staining groups showed 1597 differentially expressed genes, 1046 of which were down-regulated and 551 expression up regulated by.GO analysis. The expression of the protein encoding gene on the postsynaptic density was significantly different, and the KEGG pathway analysis of LTP and LTD was a significant difference (and the P value was the smallest). The gene of CaMK II, Gria1, Gria2, Grin1, Itpr1, Grm1, PLC beta 4 gene.6 in the postsynaptic density is consistent with the results of the gene chip 4, CaMK II, Gria1, Gria2, and beta 4 After exposure to subchronic arsenic, the expression of CaMK II protein and the expression of phosphorylated protein in the cerebellum of mice decreased (P0.05) and phosphorylated Raf and ERK1/2 protein expression decreased (P0.05) after subchronic arsenic exposure. Conclusion 1, after subchronic arsenic exposure, arsenic was accumulated in the brain tissue of mice, showing a dose-response relationship with.2 and subchronic arsenic exposure. 1 The morphological changes of the cerebellum, the subchronic arsenic exposure damage the learning and memory function.3, the subchronic arsenic exposure interferes with the expression of.4 in the cerebellar learning and memory function related genes, and the subchronic arsenic exposure causes the PSD function gene NR1, Gria1, Gria2, mGluR1, Itpr1, PLC beta 4 and CaMK II in the cerebellar cerebellum, and the PSD thickness of the cerebellar tissue changes.5, Subchronic arsenic exposure inhibits activity of CaMK II and its downstream signaling pathway in cerebellum, which may be an important mechanism of arsenic neurotoxicity.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R114

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1 ;Modulation of Behavior and Glutamate Receptor mRNA Expression in Rats after Sub-chronic Administration of Benzo(a)pyrene[J];Biomedical and Environmental Sciences;2011年04期

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本文編號(hào)：2045740