亚慢性砷暴露对小脑的神经毒作用及机制研究

发布时间：2018-06-20 21:32

本文选题：砷 + 小脑　；参考：《大连医科大学》2014年博士论文

【摘要】：背景及目的砷(Arsenic, As)是一种类金属,以无机砷和有机砷两种形态在自然界广泛分布。慢性砷中毒已成为影响数百万人健康的世界性公共卫生问题。在砷污染地区,饮用水或地下水中砷浓度达到0.25-2.1 ppm,污染严重的地方砷浓度已超过3.0ppm。孟加拉国、印度和中国是全世界受危害人口最多、病区面积最大、病情最严重的国家。我国饮水型砷中毒病区高砷暴露人群接近300万,且不包括由于燃煤引起的燃煤型地方性砷中毒。砷暴露对健康的危害是多方面的,慢性砷中毒可致引发多器官的组织学与功能上的异常改变,包括呼吸道、消化道、肝脏、肾脏、皮肤组织、免疫系统以及神经系统。神经细胞具有不能再生的特点,与其他组织比较神经细胞对毒物的毒性作用更为敏感,因此砷暴露对神经系统的毒性倍受关注。一些研究报道,急慢性砷暴露儿童会出现与神经系统损伤有关后遗症。由于脑功能受到抑制,从而导致神经组织发育障碍和智力低下,例如出现运动、认知、心理、感觉和语言等功能障碍。对孟加拉国砷含量偏高地区进行的两项流行病学调查显示,儿童水砷暴露与智力降低密切相关,与染砷动物实验结果一致。提示,砷中毒影响青少年学习记忆能力。然而,砷暴露影响学习记忆能力的机理目前尚不十分清楚。动物试验结果表明,砷暴露可导致小鼠小脑中砷含量呈剂量依赖性增高,且损伤小脑神经元,影响小脑功能。这些结果提示,小脑可能是砷毒性作用的靶器官之一。研究显示：小脑不仅负责调解肌张力、保持身体平衡和协调精细运动,而且参与认知功能的调节,包括学习能力、注意集中力、语言能力等。上述动物试验结果和文献报道表明,损伤性影响小脑功能可能与砷暴露导致的学习记忆能力下降有关。有研究报道铅、铝、氟等暴露可导致学习记忆能力下降。而且已证实铅、铝、氟的神经毒性与调控学习记忆机制相关蛋白的编码基因表达变化有关。近年来,越来越多的学者开始把小脑作为神经毒物靶器官开展研究。那么,砷的神经毒作用机制是否与小脑学习记忆相关的基因表达紊乱有关呢,引起我们极大兴趣。基因芯片技术因具有快速、准确、同时分析大量基因信息的特点,被广泛运用于生命科学领域。采用基因芯片技术研究在环境毒物刺激条件下的大量基因差异表达情况,可揭示神经毒性相关的分子生物机制。运用基因芯片技术分析砷暴露所致小脑基因表达谱变化,对筛选砷所致神经毒性相关基因及其探讨可能机制有着重要的作用。本研究采用成年小鼠进行饮水砷染毒,并观察亚慢性砷暴露对小鼠小脑学习记忆能力的影响；采用基因芯片技术筛选出砷暴露组与对照组小鼠小脑的差异表达基因,经通路分析发现长时程记忆等相关通路受到影响；在筛选出的差异表达基因中,发现突触后致密体功能基因表达受到影响,包括离子型／膜型谷氨酸受体、IP3受体、激酶CaMKⅡ、PLCβ4等；由于这些基因在长时程记忆相关通路中起到关键的作用,引起我们极大兴趣,我们观察了小脑突触后致密体超微结构,且对这些基因的表达进行验证。并检测突触后致密体关键酶CaMKⅡ的蛋白表达水平,且对CaMKⅡ下游长时程记忆相关通路的关键蛋白进行检测。为阐明砷致小脑学习记忆功能损害的分子机制以及防治砷中毒提供依据。方法1、选用月龄为一个月的健康昆明小鼠64只,体重22.3-26.4 g,在标准动物房内饲养。对照组小鼠饮用灭菌自来水,实验组小鼠分别饮用1 ppm、2 ppm和4 ppm三氧化二砷水溶液。小鼠饮用水每两天更换一次,染毒时间为2个月。2、测定脑砷含量：采用电感耦合等离子体质谱(ICP-MS)测量方法。3、HE染色观察小脑组织形态学变化。4、检测小鼠学习记忆能力和小脑运动性学习记忆能力：分别采用Morris水迷宫定向航行试验和瞬膜条件反射试验。5、小鼠小脑组织基因芯片检测,差异表达基因分析。6、检测亚慢性砷暴露对小鼠小脑突触后致密体基因表达影响。采用real time-PCR(RT-PCR)检测小脑NMDA受体亚基NR1、AMPA受体亚基GluR1和GluR2、mGlu受体亚基mGluR1、IP3受体亚基IP3R1、激酶CaMKⅡ、PLCβ4基因表达。7、透射电镜观察小鼠小脑组织突触形态结构的变化。8、检测小脑CaMKⅡ及下游通路关键蛋白表达变化。采用免疫印迹法(Western-blot)检测小鼠小脑PSD中CaMKⅡ蛋白表达和磷酸化水平；检测小鼠小脑组织Ras、Raf、ERK1/2的磷酸化水平。结果1、实验期间,各组小鼠一般情况良好。随饲养时间延长,小鼠体重增加。从第36天开始,4 ppm剂量组小鼠体重增幅减慢,与对照组和1 ppm染砷组相比,差异有统计学(P0.05)。这种差异一直维持到实验结束。在染毒末期,2 ppm剂量组小鼠体重增幅减慢,与对照组和1 ppm染砷组相比,差异有统计学意义(P0.05)。2、各实验组小鼠小脑砷含量均显著高于对照组,差异有统计学意义(P0.05)且呈剂量依赖关系。3、本研究高剂量染砷组中,小脑分子细胞层胞浆染色不均匀,纤维分布不均,细胞浆淡染,细胞皱缩。浦肯野细胞层,小脑组织浦肯野细胞层胞浆染色淡,纤维(粉染区)变大,且不均匀,细胞胞浆淡染,胞核皱缩；颗粒细胞层细胞数量减少。4、亚慢性砷暴露对小鼠运动性学习记忆能力的影响Morris水迷宫结果：对小鼠采用As203染毒60天后,与对照组比较：2 ppm和4 ppm As2O3染毒组小鼠的逃避潜伏期显著增长(P0.05),并且逃避潜伏期随着染毒剂量的增加而增长。瞬膜条件反射结果提示：As203染毒60天后,4 ppm As2O3染毒小鼠的瞬膜条件反射与对照组比较呈显著降低(P0.05)。5、基因芯片结果显示,与对照组小鼠小脑基因表达谱比较,4 ppm染砷组显示1597个差异表达基因,其中1046个表达下调,551个表达上调。GO分析提示,突触后致密体上蛋白编码基因的表达有显著性差异,KEGG通路分析LTP和LTD为有显著性差异(且P值最小)的通路。位于突触后致密体并参与LTP/LTD的基因为：CaMKⅡ、Gria1、Gria2、Grin1、Itpr1、Grm1、PLCβ4基因。6、与基因芯片结果一致,CaMKⅡ、Gria1、Gria2、Grin1、Itpr1、Grm1、PLCβ4的mRNA表达下降。7、透射电镜观察发现,As203染毒60天后,2 ppm和4 ppm As2O3染毒小鼠的突触后致密体厚度与对照组比较显著变薄(P0.05)。8、亚慢性砷暴露后,小鼠小脑CaMKⅡ 蛋白表达及磷酸化状态蛋白表达都下降(P0.05),磷酸化Raf, ERK1/2蛋白表达下降(P0.05)。结论1、亚慢性砷暴露后,砷在小鼠脑组织中蓄积,呈剂量反应关系。2、亚慢性砷暴露导致小脑组织形态学异常改变,亚慢性砷暴露损害学习记忆功能。3、亚慢性砷暴露干扰小脑学习记忆功能相关基因表达。4、亚慢性砷暴露使小鼠小脑PSD功能基因NR1、Gria1、Gria2、mGluR1、 Itpr1、PLCβ4、CaMKⅡ表达显著异常；导致小脑组织PSD厚度改变。5、亚慢性砷暴露抑制小鼠小脑CaMKⅡ及其下游信号通路活性可能是砷神经毒作用的一个重要机制。
[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.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

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