铅致阿尔茨海默症样变神经毒性及对相关通路蛋白表达的影响
发布时间：2018-08-28 20:45
【摘要】：铅是一种常见的神经毒物,对神经系统具有异常亲和力,可诱发严重的神经功能障碍和学习认知功能损伤。学习记忆和认知是高级中枢神经系统功能的基本体现。海马不仅是学习记忆发生与认知功能塑形的关键场所,也是铅神经毒性作用的主要靶位点。胰岛素/Pi3k/Akt和MAPKs信号通路的活化表达是细胞增殖、分化、凋亡、迁移等过程发生和发展的分子基础,其表达紊乱或活化异常可导致一系列疾病状态及病理表现的发生。阿尔茨海默症(Alzheimer‘s disease,AD)是一种原发型进行性神经退行性疾病,其发病受环境和遗传因素的共同调节。进行性认知功能障碍,及学习记忆能力损伤是AD的典型临床症状,该过程中常伴随Tau蛋白过磷酸化、神经元大量凋亡、突触结构/功能退化及通路蛋白表达异常。虽然AD发病机制尚不清楚,但大量研究数据提示,Aβ表达异常和蓄积可能是各种原因诱导AD发病的共同通路。近期研究发现,AD可能并不属于单纯的老年型疾病,其发病亦具有一定的胚胎源性,这符合成人疾病的胚胎基础假说(Fe BAD)的论述。已知孕哺期铅暴露会诱发严重影响子代中枢神经系统的正常发育;且人群研究结果证实发育早期铅暴露与儿童智力损伤,空间学习记忆能力障碍、认知和注意力下降具有强相关性,但该机制尚不十分清楚。本研究拟运用小鼠孕哺期铅暴露模型和PC12细胞染毒模型,检测孕早期铅暴露对子代小鼠血铅和海马铅含量、海马Aβ相关蛋白(IDE)和学习记忆相关蛋白(NGF)表达的影响;结合体外实验,观察铅暴露对神经细胞生长发育的影响,探讨该过程中可能涉及的AD相关蛋白,神经发育相关因子及MAPKs信号通路蛋白表达变化,对铅的细胞和神经毒性进行阐述和分析,为AD发病机理的探讨及神经功能修复提供理论依据。目的1.通过构建铅中毒动物模型,比较孕哺期铅暴露对仔鼠学习记忆能力的影响,检测各浓度铅暴露组海马IDE和NGF的差异表达,评估孕哺期母体铅暴露对子代小鼠神经元发育和功能表达的影响,进而说明铅的致AD样病变作用。2.通过构建体外细胞染毒模型,分析对比不同剂量及不同时间铅暴露对AD相关细胞因子,神经发育相关因子和胞内信号蛋白表达的影响,探索铅暴露对Aβ及其衍生物表达的影响,说明铅致AD样病变作用并探讨其对信号传导通路表达的影响,为AD的防治和铅毒性的干预治疗提供线索。材料与方法1研究对象动物模型:将怀孕SPF级昆明小鼠随机分为4组,每组10只,1个对照组及3个铅暴露组。孕鼠自怀孕第1d起(E0)至仔鼠断乳时,分别给予醋酸铅含量为0%(对照组)、0.1%(低剂量组)、0.2%(中剂量组)和0.5%(高剂量组)的去离子饮用水。仔鼠出生后仍由母鼠喂养照料至PND21。细胞株:选用褐家鼠肾上腺嗜铬细胞瘤PC12细胞株作为实验对象,经分化处理后,分别给予终浓度为0μM、20μM、100μM和500μM的醋酸铅染毒。2.方法2.1动物实验2.1.1采用Z-5000石墨炉原子吸收光谱仪测定仔鼠血铅和海马铅浓度。2.1.2采用Morris水迷宫实验评价仔鼠的学习记忆能力。2.1.3采用Western Blot检测IDE和NGF在不同剂量铅暴露组中的表达量。分别采用免疫组化和免疫荧光技术对IDE和NGF在海马组织中的分布和表达进行了描述(对照组和高剂量组)。2.2细胞实验2.2.1采用MTT法检测不同暴露浓度及暴露时间对细胞活性的影响。2.2.2采用Western Blot技术检测AD相关蛋白(Aβ和Aβ寡聚体),神经发育相关蛋白(IGF1/IGF1R)及胞内信号通路相关蛋白(IR、p-Akt、IDE、ERK1/2、JNK1/2/3、P38)蛋白表达的改变,采用实时定量PCR分析APP、INSR、IDE和IGF1/IGF1R基因m RNA的表达。3.统计分析实验使用SPSS 21.0软件包(SPSS Inc,USA)进行统计分析,两组数据比较采用独立样本t检验,多组数据间比较采用单因素方差分析,两两比较使用Bonferroni检验法,若不满足正态分布,则使用秩和检验,若数据满足正态分布则以均数±标准差(x±s)表示,检验水平为α=0.05。结果1.动物实验结果1.1孕哺期母体铅暴露对仔鼠血铅、海马铅含量及其学习记忆能力的影响不同剂量孕哺期铅暴露后,PND21仔鼠血铅和海马铅含量显著高于对照组(P0.05)。Morris水迷宫结果显示,中、高暴露剂量组仔鼠的逃避潜伏期和错误次数均显著高于对照组(P0.05)。1.2.孕哺期母体铅暴露对仔鼠海马组织IDE和NGF蛋白表达的影响1.2.1 Western Blot结果显示,各铅暴露组仔鼠海马IDE和NGFβ蛋白表达水平较对照组均明显降低(P0.05)。1.2.2免疫组化结果显示,高剂量铅暴露组仔鼠海马CA1区NGF免疫组化阳性反应物的平均灰度值明显低于对照组(P0.05)。1.2.3免疫荧光实验结果显示,高剂量铅暴露组仔鼠海马神经元活性剂IDE免疫荧光抗体的平均光密度均显著低于对照组(P0.05)。2.细胞实验结果1.醋酸铅处理对PC12细胞增殖的影响根据细胞的数量变化及形态学改变最终选择0μM、20μM、100μM和500μM醋酸铅暴露浓度及12h、24h和72h来开展后续研究。2.醋酸铅处理对PC12细胞Aβ和Aβ寡聚体表达的影响不同浓度醋酸铅暴露12h后,500μM染铅组PC12细胞Aβ的表达低于对照组(P0.05);Aβ寡聚体蛋白在20μM染铅组细胞中的表达低于对照组(P0.05),100μM和500μM染铅组PC12细胞Aβ蛋白的表达水平高于对照组(P0.05)。染毒24h后,各染铅组细胞Aβ的表达水平均低于对照组(P0.05);Aβ寡聚体蛋白在各染铅组细胞中的表达无明显改变(P0.05)。染毒72h后,Aβ蛋白在100μM和500μM染铅组细胞中的表达低于对照组(P0.05);Aβ寡聚体蛋白在各染铅组细胞中的表达均高于对照组(P0.05)。3.醋酸铅处理对PC12细胞IGF1/IGF1R表达的影响醋酸铅暴露12h后,20μM染铅组PC12细胞IGF1的表达高于对照组(P0.05),IGF1和IGF1R蛋白在100μM和500μM染铅组细胞中的表达均低于对照组(P0.05)。染毒24h后,IGF1蛋白在各染铅组细胞中的表达均低于对照组(P0.05);IGF1R蛋白在20μM和500μM染铅组细胞中的表达低于对照组(P0.05)。染毒72h后,IGF1蛋白在各染铅组细胞中的表达均低于对照组(P0.05);100μM和500μM染铅组细胞IGF1R的表达低于对照组(P0.05)。4.醋酸铅处理对PC12细胞IR、p-Akt、IDE表达的影响染毒12h后,20μM染铅细胞IR的表达低于对照组(P0.05),100μM染铅组细胞IR和p-Akt蛋白表达水平均高于对照组(P0.05);500μM染铅组细胞p-Akt的表达低于对照组(P0.05);500μM染铅组细胞IDE的表达低于对照组(P0.05)。染毒24h后,20μM及100μM染铅组细胞IR的表达低于对照组(P0.05);p-Akt蛋白在20μM和100μM染铅组细胞中的表达显著高于对照组(P0.05),500μM染铅组细胞p-Akt的表达量则明显低于对照组(P0.05);IDE蛋白在各染铅组细胞中的表达均低于对照组(P0.05)。染毒72h后,IR蛋白在20μM和500μM染铅组细胞中的表达低于对照组(P0.05);p-Akt蛋白在500μM染铅组细胞中的表达低于对照组(P0.05);100μM及500μM染铅组细胞IDE的表达低于对照组(P0.05)。5.醋酸铅处理对PC12细胞MAPK信号通路相关蛋白(ERK1/2、JNK1/2/3、P38)表达的影响不同浓度醋酸铅处理12h后,与对照组比较,各染铅组细胞ERK1的表达增高,而ERK2和P38蛋白表达降低(P0.05);500μM染铅组细胞JNK1的表达低于对照组(P0.05);20μM及100μM染铅组细胞JNK2的表达高于对照组(P0.05);100μM及500μM染铅组细胞JNK3的表达低于对照组(P0.05)。染毒24h后,与对照组比较,20μM染铅组细胞ERK1表达显著升高,而500μM染铅组细胞ERK1蛋白表达降低(P0.05);ERK2蛋白在各染铅组细胞中的表达均低于对照组(P0.05);100μM及500μM染铅组细胞JNK1的表达低于对照组(P0.05);500μM染铅组细胞JNK2蛋白的表达低于对照组(P0.05);20μM及500μM染铅组细胞JNK3的表达低于对照组(P0.05);P38蛋白在100μM和500μM染铅组细胞中的表达低于对照组(P0.05)。染毒72h后,与对照组比较,各染铅组细胞ERK2及JNK1蛋白的表达均降低(P0.05);500μM染铅组细胞ERK1的表达低于对照组(P0.05);100μM染铅组细胞JNK2的表达高于对照组(P0.05),500μM染铅组细胞JNK2的表达低于对照组(P0.05);JNK3蛋白在100μM及500μM染铅组细胞中的表达均低于对照组(P0.05);100μM及500μM染铅组细胞P38的表达低于对照组(P0.05)。6.醋酸铅对PC12细胞APP、INSR、AKT、IDE和IGF1/IGF1R转录水平的影响PCR结果显示,醋酸铅处理12h后,APP m RNA在各染铅组细胞中的表达无明显改变(P0.05);20μM染铅组细胞INSR m RNA的表达低于对照组(P0.05);各染铅组细胞AKT m RNA的表达均低于对照组(P0.05);各实验组细胞IDE m RNA的表达无显著差异(P0.05);20μM和100μM染铅组细胞IGF1/IGF1R m RNA的表达均低于对照组(P0.05)。染毒24h后,20μM及100μM染铅组细胞APP m RNA的表达高于对照组(P0.05);100μM染铅组细胞INSR m RNA的表达低于对照组(P0.05);20μM及100μM染铅组细胞AKT m RNA的表达低于对照组(P0.05);20μM染铅组细胞IDE m RNA的表达低于对照组(P0.05);100μM染铅组细胞IGF1 m RNA的表达低于对照组(P0.05),各实验组细胞IGF1R m RNA的表达无明显差异(P0.05)。染毒72h后,20μM及100μM染铅组细胞APP m RNA的表达均低于对照组(P0.05);仅20μM染铅组细胞INSR m RNA的表达低于对照组(P0.05);AKT m RNA在20μM及100μM染铅组细胞中的表达低于对照组(P0.05);IDE m RNA在各实验组中的表达无明显改变(P0.05);20μM和100μM醋酸染铅组细胞IGF1/IGF1R m RNA的表达均低于对照组(P0.05)。结论1.孕哺期母体铅暴露可导致仔鼠血铅和海马铅浓度明显升高,抑制Aβ降解酶IDE及神经生长因子NGF的表达,损伤仔鼠的空间学习能力,该损伤效应呈暴露浓度依赖性。提示子代学习记忆能力障碍、脑AD样神经退行性变与孕哺期母体铅暴露密切相关。2.细胞染毒实验结果与动物研究结果相一致。醋酸铅暴露会对PC12细胞AD相关蛋白表达产生影响,该过程可能涉及胞内信号通路蛋白的异常表达。铅暴露在抑制ERK2、JNK1/3及P38蛋白表达的同时,刺激了ERK1和JNK2 MAPKs的表达。说明铅暴露可通过诱发信号通路表达异常来诱导AD样病变的产生。
[Abstract]:Lead is a common neurotoxic substance with abnormal affinity to the nervous system, which can induce severe neurological dysfunction and impairment of learning and cognition.Learning, memory and cognition are the basic manifestations of advanced central nervous system function. Activated expression of insulin/Pi3k/Akt and MAPKs signaling pathways is the molecular basis of cell proliferation, differentiation, apoptosis, migration and other processes. Abnormal expression or activation of insulin/Pi3k/Akt signaling pathways may lead to a series of disease states and pathological manifestations. Alzheimer's disease (AD) is a primary type of Alzheimer's disease. Progressive neurodegenerative diseases are regulated by both environmental and genetic factors. Progressive cognitive impairment and impairment of learning and memory are typical clinical symptoms of AD. This process is often accompanied by hyperphosphorylation of Tau protein, apoptosis of neurons, degeneration of synaptic structure/function and abnormal expression of pathway protein. A large number of data suggest that the abnormal expression and accumulation of Abeta may be the common pathway leading to the onset of AD. Recent studies have found that AD may not be a simple senile disease, but also has embryogenic origin, which is consistent with the embryonic basis hypothesis (Fe BAD) of adult diseases. Lead exposure can seriously affect the normal development of the central nervous system in offspring; and the results of population studies have confirmed that early lead exposure is strongly associated with mental impairment, spatial learning and memory impairment, cognitive and attention loss in children, but the mechanism is not very clear. 2 cell toxicity model was used to detect the effects of lead exposure in early pregnancy on blood lead and hippocampal lead content, hippocampal A beta associated protein (IDE) and learning and memory related protein (NGF) expression in mice; combined with in vitro experiments, the effects of lead exposure on the growth and development of neural cells were observed, and the possible AD related proteins and neurodevelopmental related factors were discussed. Objective 1. To compare the effects of lead exposure during pregnancy and lactation on the learning and memory abilities of offspring and to detect the IDE in hippocampus of rats exposed to lead at different concentrations. To evaluate the effect of maternal lead exposure during pregnancy and lactation on neuronal development and functional expression in offspring of mice, and then to explain the role of lead-induced AD-like lesions. 2. By constructing an in vitro cell toxicity model, the AD-related cytokines, neurodevelopment-related factors and intracellular signal eggs were analyzed and compared in different dose and time of lead exposure. Materials and Methods 1 Animal model of pregnant SPF Kunming mice were randomly divided into 4 groups, 10 mice in each group. From the first day of pregnancy (E0) to weaning, pregnant mice were given deionized drinking water containing 0% (control group), 0.1% (low dose group), 0.2% (middle dose group) and 0.5% (high dose group) of lead. The offspring were still fed and cared for by the mother mice until PND21. Cell strain: Rattus norvegicus adrenal chromaffin cells were selected. Methods 2.1 Animal experiment 2.1.1 Determination of blood lead and hippocampal lead concentration in offspring by Z-5000 graphite furnace atomic absorption spectrometry 2.1.2 Morris water maze test was used to evaluate the learning and memory ability of offspring. Western Blot was used to detect the expression of IDE and NGF in different lead exposure groups. Immunohistochemistry and immunofluorescence were used to describe the distribution and expression of IDE and NGF in hippocampus (control group and high dose group). 2.2.1 Cell experiment 2.2. The expression of AD-related proteins (A beta and A beta oligomers), nerve development-related proteins (IGF1/IGF1R) and intracellular signaling pathway-related proteins (IR, p-Akt, IDE, ERK1/2, JNK1/2/3, P38) were detected by Western Blot. The expression of M RNA in APP, INSR, IDE and IGF1/IGF1R genes was analyzed by real-time quantitative PCR. SPSS 21.0 software package (SPSS Inc, USA) was used for statistical analysis. Independent sample t test was used for comparison between the two groups of data. One-way ANOVA was used for comparison among multiple groups of data. Bonferroni test was used for pairwise comparison. If the normal distribution was not satisfied, the rank sum test was used. If the normal distribution was satisfied, the data was expressed as mean (+s). Results 1.1 Effects of maternal lead exposure on blood lead, hippocampal lead content and learning and memory ability of offspring rats after different doses of lead exposure during pregnancy and feeding period, blood lead and hippocampal lead content of PND21 offspring was significantly higher than that of control group (P The expression of IDE and NGF beta protein in hippocampus of offspring exposed to lead during pregnancy and lactation was significantly lower than that of control group (P 0.05). 1.2. The results of Western Blot showed that the expression of IDE and NGF beta protein in hippocampus of offspring exposed to lead was significantly lower than that of control group (P 0.05). The average gray value of NGF immunohistochemical positive reactants in CA1 area of hippocampus of rats exposed to lead was significantly lower than that of control group (P 0.05). 1.2.3 immunofluorescence test showed that the average optical density of IDE immunofluorescent antibody in hippocampus of rats exposed to lead was significantly lower than that of control group (P 0.05). The effects of lead acetate treatment on the proliferation of PC12 cells were studied according to the number and morphological changes of PC12 cells. 2. Effects of lead acetate treatment on the expression of A beta and A beta oligomers in PC12 cells exposed to lead acetate at different concentrations 12 hours after exposure to lead acetate at different concentrations The expression of Abeta oligomer protein was lower than that of the control group (P 0.05); the expression of Abeta oligomer protein was lower in the lead-exposed group (P 0.05); the expression of Abeta protein in PC12 cells was higher in the lead-exposed group (P 0.05) than that in the control group (P 0.05). The expression of A-beta protein in PC12 cells was lower than that in control group 72 hours after exposure (P 0.05). The expression of A-beta oligomer protein in PC12 cells was higher than that in control group (P 0.05). The expression of IGF1 in PC12 cells was higher than that in the control group (P 0.05), and the expression of IGF1 and IGF1R protein in the lead-exposed group was lower than that in the control group (P 0.05). After 72 hours of exposure, the expression of IGF1 protein was lower than that of the control group (P 0.05), and the expression of IGF1R was lower in 100 and 500 mu lead exposure groups than that of the control group (P 0.05). The expression of P-Akt protein in lead-exposed group was lower than that in control group (P 0.05); the expression of IDE in lead-exposed group was lower than that in lead-exposed group (P 0.05). 24 hours after exposure, the expression of IR in lead-exposed group was lower than that in control group (P 0.05); the expression of p-Akt protein in lead-exposed group was significantly higher than that in lead-exposed group (P 0.05). In the control group (P 0.05), the expression of p-Akt was significantly lower in the lead-exposed group than in the control group (P 0.05); the expression of IDE protein in the lead-exposed group was lower than that in the control group (P 0.05). 72 hours after exposure, the expression of IR protein in the lead-exposed group was lower than that in the control group (P 0.05); the expression of p-Akt protein in the lead-exposed group was lower than that in the lead-exposed group (P 0.05). The expression of IDE in PC12 cells treated with lead acetate at different concentrations (ERK1/2, JNK1/2/3, P38) increased, while the expression of ERK 2 and P38 decreased (P 0.05). The expression of JNK1 in lead-exposed group was lower than that in control group (P 0.05); the expression of JNK2 in lead-exposed group was higher than that in control group (P 0.05); the expression of JNK3 in lead-exposed group was lower than that in lead-exposed group (P 0.05); the expression of ERK1 in lead-exposed group was significantly higher than that in lead-exposed group (P 0.05). The expression of ERK2 protein was lower than that of control group (P 0.05); the expression of JNK1 protein was lower in 100 and 500 mu lead group (P 0.05); the expression of JNK2 protein in 500 mu lead group was lower than that of control group (P 0.05); the expression of JNK3 protein in 20 and 500 mu lead group was lower than that of control group (P 0.05); Compared with the control group, the expression of ERK2 and JNK1 protein decreased 72 hours after exposure (P 0.05); the expression of ERK1 was lower in the 500 mu lead group than in the control group (P 0.05); the expression of JNK2 in the 100 mu lead group was higher than that in the control group (P 0.05); and the expression of JNK2 in the 500 mu lead group was higher than that in the 500 mu lead group (P 0.05). The expression of JNK3 protein was lower than that of control group (P 0.05); the expression of P38 protein was lower in 100 and 500 mu lead exposed group (P 0.05); the expression of P38 in 100 and 500 mu lead exposed group was lower than that of control group (P 0.05). 6. There was no significant change in the expression of A in all lead-exposed groups (P 0.05); the expression of INSR m RNA in lead-exposed group was lower than that in control group (P 0.05); the expression of AKT m RNA in all lead-exposed groups was lower than that in control group (P 0.05); there was no significant difference in the expression of IDE M RNA in all experimental groups (P 0.05); the expression of IGF1/IGF1R m RNA in lead-exposed group was lower than that in lead-exposed group (P 0.05). 24 hours after exposure, the expression of APP m RNA in lead exposed group was higher than that in control group (P 0.05); the expression of INSR m RNA in lead exposed group was lower than that in control group (P 0.05); the expression of AKT m RNA in lead exposed group was lower than that in control group (P 0.05); the expression of IDE m RNA in lead exposed group was lower than that in control group (P 0.05). After 72 hours of exposure, the expression of APP m RNA in the 20 and 100 mu lead groups was lower than that in the control group (P 0.05), and the expression of INSR m RNA in the 20 and 100 mu lead group was lower than that in the control group (P 0.05). The expression of IGF1/IGF1R m RNA in lead exposed group was lower than that in control group (P 0.05); the expression of IDE-m RNA was not significantly changed in each experimental group (P 0.05); the expression of IGF1/IGF1R-m RNA in lead exposed group was lower than that in control group (P 0.05). Conclusion 1. Lead exposure in maternal body during pregnancy and feeding period could significantly increase the blood lead and hippocampal lead concentration and inhibit the degradation of A beta. The expression of enzyme IDE and NGF impaired the spatial learning ability of offspring in a dose-dependent manner, suggesting that offspring'learning and memory impairment and brain AD-like neurodegeneration were closely related to maternal lead exposure during gestation and lactation. 2. The results of cell toxicity test were consistent with animal studies. The expression of AD-related proteins in cells is affected by abnormal expression of intracellular signaling pathway proteins. Lead exposure is inhibited
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16
，
本文编号：2210568
[Abstract]:Lead is a common neurotoxic substance with abnormal affinity to the nervous system, which can induce severe neurological dysfunction and impairment of learning and cognition.Learning, memory and cognition are the basic manifestations of advanced central nervous system function. Activated expression of insulin/Pi3k/Akt and MAPKs signaling pathways is the molecular basis of cell proliferation, differentiation, apoptosis, migration and other processes. Abnormal expression or activation of insulin/Pi3k/Akt signaling pathways may lead to a series of disease states and pathological manifestations. Alzheimer's disease (AD) is a primary type of Alzheimer's disease. Progressive neurodegenerative diseases are regulated by both environmental and genetic factors. Progressive cognitive impairment and impairment of learning and memory are typical clinical symptoms of AD. This process is often accompanied by hyperphosphorylation of Tau protein, apoptosis of neurons, degeneration of synaptic structure/function and abnormal expression of pathway protein. A large number of data suggest that the abnormal expression and accumulation of Abeta may be the common pathway leading to the onset of AD. Recent studies have found that AD may not be a simple senile disease, but also has embryogenic origin, which is consistent with the embryonic basis hypothesis (Fe BAD) of adult diseases. Lead exposure can seriously affect the normal development of the central nervous system in offspring; and the results of population studies have confirmed that early lead exposure is strongly associated with mental impairment, spatial learning and memory impairment, cognitive and attention loss in children, but the mechanism is not very clear. 2 cell toxicity model was used to detect the effects of lead exposure in early pregnancy on blood lead and hippocampal lead content, hippocampal A beta associated protein (IDE) and learning and memory related protein (NGF) expression in mice; combined with in vitro experiments, the effects of lead exposure on the growth and development of neural cells were observed, and the possible AD related proteins and neurodevelopmental related factors were discussed. Objective 1. To compare the effects of lead exposure during pregnancy and lactation on the learning and memory abilities of offspring and to detect the IDE in hippocampus of rats exposed to lead at different concentrations. To evaluate the effect of maternal lead exposure during pregnancy and lactation on neuronal development and functional expression in offspring of mice, and then to explain the role of lead-induced AD-like lesions. 2. By constructing an in vitro cell toxicity model, the AD-related cytokines, neurodevelopment-related factors and intracellular signal eggs were analyzed and compared in different dose and time of lead exposure. Materials and Methods 1 Animal model of pregnant SPF Kunming mice were randomly divided into 4 groups, 10 mice in each group. From the first day of pregnancy (E0) to weaning, pregnant mice were given deionized drinking water containing 0% (control group), 0.1% (low dose group), 0.2% (middle dose group) and 0.5% (high dose group) of lead. The offspring were still fed and cared for by the mother mice until PND21. Cell strain: Rattus norvegicus adrenal chromaffin cells were selected. Methods 2.1 Animal experiment 2.1.1 Determination of blood lead and hippocampal lead concentration in offspring by Z-5000 graphite furnace atomic absorption spectrometry 2.1.2 Morris water maze test was used to evaluate the learning and memory ability of offspring. Western Blot was used to detect the expression of IDE and NGF in different lead exposure groups. Immunohistochemistry and immunofluorescence were used to describe the distribution and expression of IDE and NGF in hippocampus (control group and high dose group). 2.2.1 Cell experiment 2.2. The expression of AD-related proteins (A beta and A beta oligomers), nerve development-related proteins (IGF1/IGF1R) and intracellular signaling pathway-related proteins (IR, p-Akt, IDE, ERK1/2, JNK1/2/3, P38) were detected by Western Blot. The expression of M RNA in APP, INSR, IDE and IGF1/IGF1R genes was analyzed by real-time quantitative PCR. SPSS 21.0 software package (SPSS Inc, USA) was used for statistical analysis. Independent sample t test was used for comparison between the two groups of data. One-way ANOVA was used for comparison among multiple groups of data. Bonferroni test was used for pairwise comparison. If the normal distribution was not satisfied, the rank sum test was used. If the normal distribution was satisfied, the data was expressed as mean (+s). Results 1.1 Effects of maternal lead exposure on blood lead, hippocampal lead content and learning and memory ability of offspring rats after different doses of lead exposure during pregnancy and feeding period, blood lead and hippocampal lead content of PND21 offspring was significantly higher than that of control group (P The expression of IDE and NGF beta protein in hippocampus of offspring exposed to lead during pregnancy and lactation was significantly lower than that of control group (P 0.05). 1.2. The results of Western Blot showed that the expression of IDE and NGF beta protein in hippocampus of offspring exposed to lead was significantly lower than that of control group (P 0.05). The average gray value of NGF immunohistochemical positive reactants in CA1 area of hippocampus of rats exposed to lead was significantly lower than that of control group (P 0.05). 1.2.3 immunofluorescence test showed that the average optical density of IDE immunofluorescent antibody in hippocampus of rats exposed to lead was significantly lower than that of control group (P 0.05). The effects of lead acetate treatment on the proliferation of PC12 cells were studied according to the number and morphological changes of PC12 cells. 2. Effects of lead acetate treatment on the expression of A beta and A beta oligomers in PC12 cells exposed to lead acetate at different concentrations 12 hours after exposure to lead acetate at different concentrations The expression of Abeta oligomer protein was lower than that of the control group (P 0.05); the expression of Abeta oligomer protein was lower in the lead-exposed group (P 0.05); the expression of Abeta protein in PC12 cells was higher in the lead-exposed group (P 0.05) than that in the control group (P 0.05). The expression of A-beta protein in PC12 cells was lower than that in control group 72 hours after exposure (P 0.05). The expression of A-beta oligomer protein in PC12 cells was higher than that in control group (P 0.05). The expression of IGF1 in PC12 cells was higher than that in the control group (P 0.05), and the expression of IGF1 and IGF1R protein in the lead-exposed group was lower than that in the control group (P 0.05). After 72 hours of exposure, the expression of IGF1 protein was lower than that of the control group (P 0.05), and the expression of IGF1R was lower in 100 and 500 mu lead exposure groups than that of the control group (P 0.05). The expression of P-Akt protein in lead-exposed group was lower than that in control group (P 0.05); the expression of IDE in lead-exposed group was lower than that in lead-exposed group (P 0.05). 24 hours after exposure, the expression of IR in lead-exposed group was lower than that in control group (P 0.05); the expression of p-Akt protein in lead-exposed group was significantly higher than that in lead-exposed group (P 0.05). In the control group (P 0.05), the expression of p-Akt was significantly lower in the lead-exposed group than in the control group (P 0.05); the expression of IDE protein in the lead-exposed group was lower than that in the control group (P 0.05). 72 hours after exposure, the expression of IR protein in the lead-exposed group was lower than that in the control group (P 0.05); the expression of p-Akt protein in the lead-exposed group was lower than that in the lead-exposed group (P 0.05). The expression of IDE in PC12 cells treated with lead acetate at different concentrations (ERK1/2, JNK1/2/3, P38) increased, while the expression of ERK 2 and P38 decreased (P 0.05). The expression of JNK1 in lead-exposed group was lower than that in control group (P 0.05); the expression of JNK2 in lead-exposed group was higher than that in control group (P 0.05); the expression of JNK3 in lead-exposed group was lower than that in lead-exposed group (P 0.05); the expression of ERK1 in lead-exposed group was significantly higher than that in lead-exposed group (P 0.05). The expression of ERK2 protein was lower than that of control group (P 0.05); the expression of JNK1 protein was lower in 100 and 500 mu lead group (P 0.05); the expression of JNK2 protein in 500 mu lead group was lower than that of control group (P 0.05); the expression of JNK3 protein in 20 and 500 mu lead group was lower than that of control group (P 0.05); Compared with the control group, the expression of ERK2 and JNK1 protein decreased 72 hours after exposure (P 0.05); the expression of ERK1 was lower in the 500 mu lead group than in the control group (P 0.05); the expression of JNK2 in the 100 mu lead group was higher than that in the control group (P 0.05); and the expression of JNK2 in the 500 mu lead group was higher than that in the 500 mu lead group (P 0.05). The expression of JNK3 protein was lower than that of control group (P 0.05); the expression of P38 protein was lower in 100 and 500 mu lead exposed group (P 0.05); the expression of P38 in 100 and 500 mu lead exposed group was lower than that of control group (P 0.05). 6. There was no significant change in the expression of A in all lead-exposed groups (P 0.05); the expression of INSR m RNA in lead-exposed group was lower than that in control group (P 0.05); the expression of AKT m RNA in all lead-exposed groups was lower than that in control group (P 0.05); there was no significant difference in the expression of IDE M RNA in all experimental groups (P 0.05); the expression of IGF1/IGF1R m RNA in lead-exposed group was lower than that in lead-exposed group (P 0.05). 24 hours after exposure, the expression of APP m RNA in lead exposed group was higher than that in control group (P 0.05); the expression of INSR m RNA in lead exposed group was lower than that in control group (P 0.05); the expression of AKT m RNA in lead exposed group was lower than that in control group (P 0.05); the expression of IDE m RNA in lead exposed group was lower than that in control group (P 0.05). After 72 hours of exposure, the expression of APP m RNA in the 20 and 100 mu lead groups was lower than that in the control group (P 0.05), and the expression of INSR m RNA in the 20 and 100 mu lead group was lower than that in the control group (P 0.05). The expression of IGF1/IGF1R m RNA in lead exposed group was lower than that in control group (P 0.05); the expression of IDE-m RNA was not significantly changed in each experimental group (P 0.05); the expression of IGF1/IGF1R-m RNA in lead exposed group was lower than that in control group (P 0.05). Conclusion 1. Lead exposure in maternal body during pregnancy and feeding period could significantly increase the blood lead and hippocampal lead concentration and inhibit the degradation of A beta. The expression of enzyme IDE and NGF impaired the spatial learning ability of offspring in a dose-dependent manner, suggesting that offspring'learning and memory impairment and brain AD-like neurodegeneration were closely related to maternal lead exposure during gestation and lactation. 2. The results of cell toxicity test were consistent with animal studies. The expression of AD-related proteins in cells is affected by abnormal expression of intracellular signaling pathway proteins. Lead exposure is inhibited
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16
，
本文编号：2210568
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