發(fā)布時間：2018-08-28 20:45

【摘要】：鉛是一種常見的神經毒物,對神經系統(tǒng)具有異常親和力,可誘發(fā)嚴重的神經功能障礙和學習認知功能損傷。學習記憶和認知是高級中樞神經系統(tǒng)功能的基本體現(xiàn)。海馬不僅是學習記憶發(fā)生與認知功能塑形的關鍵場所,也是鉛神經毒性作用的主要靶位點。胰島素/Pi3k/Akt和MAPKs信號通路的活化表達是細胞增殖、分化、凋亡、遷移等過程發(fā)生和發(fā)展的分子基礎,其表達紊亂或活化異�？蓪е乱幌盗屑膊顟B(tài)及病理表現(xiàn)的發(fā)生。阿爾茨海默癥(Alzheimer‘s disease,AD)是一種原發(fā)型進行性神經退行性疾病,其發(fā)病受環(huán)境和遺傳因素的共同調節(jié)。進行性認知功能障礙,及學習記憶能力損傷是AD的典型臨床癥狀,該過程中常伴隨Tau蛋白過磷酸化、神經元大量凋亡、突觸結構/功能退化及通路蛋白表達異常。雖然AD發(fā)病機制尚不清楚,但大量研究數(shù)據(jù)提示,Aβ表達異常和蓄積可能是各種原因誘導AD發(fā)病的共同通路。近期研究發(fā)現(xiàn),AD可能并不屬于單純的老年型疾病,其發(fā)病亦具有一定的胚胎源性,這符合成人疾病的胚胎基礎假說(Fe BAD)的論述。已知孕哺期鉛暴露會誘發(fā)嚴重影響子代中樞神經系統(tǒng)的正常發(fā)育;且人群研究結果證實發(fā)育早期鉛暴露與兒童智力損傷,空間學習記憶能力障礙、認知和注意力下降具有強相關性,但該機制尚不十分清楚。本研究擬運用小鼠孕哺期鉛暴露模型和PC12細胞染毒模型,檢測孕早期鉛暴露對子代小鼠血鉛和海馬鉛含量、海馬Aβ相關蛋白(IDE)和學習記憶相關蛋白(NGF)表達的影響;結合體外實驗,觀察鉛暴露對神經細胞生長發(fā)育的影響,探討該過程中可能涉及的AD相關蛋白,神經發(fā)育相關因子及MAPKs信號通路蛋白表達變化,對鉛的細胞和神經毒性進行闡述和分析,為AD發(fā)病機理的探討及神經功能修復提供理論依據(jù)。目的1.通過構建鉛中毒動物模型,比較孕哺期鉛暴露對仔鼠學習記憶能力的影響,檢測各濃度鉛暴露組海馬IDE和NGF的差異表達,評估孕哺期母體鉛暴露對子代小鼠神經元發(fā)育和功能表達的影響,進而說明鉛的致AD樣病變作用。2.通過構建體外細胞染毒模型,分析對比不同劑量及不同時間鉛暴露對AD相關細胞因子,神經發(fā)育相關因子和胞內信號蛋白表達的影響,探索鉛暴露對Aβ及其衍生物表達的影響,說明鉛致AD樣病變作用并探討其對信號傳導通路表達的影響,為AD的防治和鉛毒性的干預治療提供線索。材料與方法1研究對象動物模型:將懷孕SPF級昆明小鼠隨機分為4組,每組10只,1個對照組及3個鉛暴露組。孕鼠自懷孕第1d起(E0)至仔鼠斷乳時,分別給予醋酸鉛含量為0%(對照組)、0.1%(低劑量組)、0.2%(中劑量組)和0.5%(高劑量組)的去離子飲用水。仔鼠出生后仍由母鼠喂養(yǎng)照料至PND21。細胞株:選用褐家鼠腎上腺嗜鉻細胞瘤PC12細胞株作為實驗對象,經分化處理后,分別給予終濃度為0μM、20μM、100μM和500μM的醋酸鉛染毒。2.方法2.1動物實驗2.1.1采用Z-5000石墨爐原子吸收光譜儀測定仔鼠血鉛和海馬鉛濃度。2.1.2采用Morris水迷宮實驗評價仔鼠的學習記憶能力。2.1.3采用Western Blot檢測IDE和NGF在不同劑量鉛暴露組中的表達量。分別采用免疫組化和免疫熒光技術對IDE和NGF在海馬組織中的分布和表達進行了描述(對照組和高劑量組)。2.2細胞實驗2.2.1采用MTT法檢測不同暴露濃度及暴露時間對細胞活性的影響。2.2.2采用Western Blot技術檢測AD相關蛋白(Aβ和Aβ寡聚體),神經發(fā)育相關蛋白(IGF1/IGF1R)及胞內信號通路相關蛋白(IR、p-Akt、IDE、ERK1/2、JNK1/2/3、P38)蛋白表達的改變,采用實時定量PCR分析APP、INSR、IDE和IGF1/IGF1R基因m RNA的表達。3.統(tǒng)計分析實驗使用SPSS 21.0軟件包(SPSS Inc,USA)進行統(tǒng)計分析,兩組數(shù)據(jù)比較采用獨立樣本t檢驗,多組數(shù)據(jù)間比較采用單因素方差分析,兩兩比較使用Bonferroni檢驗法,若不滿足正態(tài)分布,則使用秩和檢驗,若數(shù)據(jù)滿足正態(tài)分布則以均數(shù)±標準差(x±s)表示,檢驗水平為α=0.05。結果1.動物實驗結果1.1孕哺期母體鉛暴露對仔鼠血鉛、海馬鉛含量及其學習記憶能力的影響不同劑量孕哺期鉛暴露后,PND21仔鼠血鉛和海馬鉛含量顯著高于對照組(P0.05)。Morris水迷宮結果顯示,中、高暴露劑量組仔鼠的逃避潛伏期和錯誤次數(shù)均顯著高于對照組(P0.05)。1.2.孕哺期母體鉛暴露對仔鼠海馬組織IDE和NGF蛋白表達的影響1.2.1 Western Blot結果顯示,各鉛暴露組仔鼠海馬IDE和NGFβ蛋白表達水平較對照組均明顯降低(P0.05)。1.2.2免疫組化結果顯示,高劑量鉛暴露組仔鼠海馬CA1區(qū)NGF免疫組化陽性反應物的平均灰度值明顯低于對照組(P0.05)。1.2.3免疫熒光實驗結果顯示,高劑量鉛暴露組仔鼠海馬神經元活性劑IDE免疫熒光抗體的平均光密度均顯著低于對照組(P0.05)。2.細胞實驗結果1.醋酸鉛處理對PC12細胞增殖的影響根據(jù)細胞的數(shù)量變化及形態(tài)學改變最終選擇0μM、20μM、100μM和500μM醋酸鉛暴露濃度及12h、24h和72h來開展后續(xù)研究。2.醋酸鉛處理對PC12細胞Aβ和Aβ寡聚體表達的影響不同濃度醋酸鉛暴露12h后,500μM染鉛組PC12細胞Aβ的表達低于對照組(P0.05);Aβ寡聚體蛋白在20μM染鉛組細胞中的表達低于對照組(P0.05),100μM和500μM染鉛組PC12細胞Aβ蛋白的表達水平高于對照組(P0.05)。染毒24h后,各染鉛組細胞Aβ的表達水平均低于對照組(P0.05);Aβ寡聚體蛋白在各染鉛組細胞中的表達無明顯改變(P0.05)。染毒72h后,Aβ蛋白在100μM和500μM染鉛組細胞中的表達低于對照組(P0.05);Aβ寡聚體蛋白在各染鉛組細胞中的表達均高于對照組(P0.05)。3.醋酸鉛處理對PC12細胞IGF1/IGF1R表達的影響醋酸鉛暴露12h后,20μM染鉛組PC12細胞IGF1的表達高于對照組(P0.05),IGF1和IGF1R蛋白在100μM和500μM染鉛組細胞中的表達均低于對照組(P0.05)。染毒24h后,IGF1蛋白在各染鉛組細胞中的表達均低于對照組(P0.05);IGF1R蛋白在20μM和500μM染鉛組細胞中的表達低于對照組(P0.05)。染毒72h后,IGF1蛋白在各染鉛組細胞中的表達均低于對照組(P0.05);100μM和500μM染鉛組細胞IGF1R的表達低于對照組(P0.05)。4.醋酸鉛處理對PC12細胞IR、p-Akt、IDE表達的影響染毒12h后,20μM染鉛細胞IR的表達低于對照組(P0.05),100μM染鉛組細胞IR和p-Akt蛋白表達水平均高于對照組(P0.05);500μM染鉛組細胞p-Akt的表達低于對照組(P0.05);500μM染鉛組細胞IDE的表達低于對照組(P0.05)。染毒24h后,20μM及100μM染鉛組細胞IR的表達低于對照組(P0.05);p-Akt蛋白在20μM和100μM染鉛組細胞中的表達顯著高于對照組(P0.05),500μM染鉛組細胞p-Akt的表達量則明顯低于對照組(P0.05);IDE蛋白在各染鉛組細胞中的表達均低于對照組(P0.05)。染毒72h后,IR蛋白在20μM和500μM染鉛組細胞中的表達低于對照組(P0.05);p-Akt蛋白在500μM染鉛組細胞中的表達低于對照組(P0.05);100μM及500μM染鉛組細胞IDE的表達低于對照組(P0.05)。5.醋酸鉛處理對PC12細胞MAPK信號通路相關蛋白(ERK1/2、JNK1/2/3、P38)表達的影響不同濃度醋酸鉛處理12h后,與對照組比較,各染鉛組細胞ERK1的表達增高,而ERK2和P38蛋白表達降低(P0.05);500μM染鉛組細胞JNK1的表達低于對照組(P0.05);20μM及100μM染鉛組細胞JNK2的表達高于對照組(P0.05);100μM及500μM染鉛組細胞JNK3的表達低于對照組(P0.05)。染毒24h后,與對照組比較,20μM染鉛組細胞ERK1表達顯著升高,而500μM染鉛組細胞ERK1蛋白表達降低(P0.05);ERK2蛋白在各染鉛組細胞中的表達均低于對照組(P0.05);100μM及500μM染鉛組細胞JNK1的表達低于對照組(P0.05);500μM染鉛組細胞JNK2蛋白的表達低于對照組(P0.05);20μM及500μM染鉛組細胞JNK3的表達低于對照組(P0.05);P38蛋白在100μM和500μM染鉛組細胞中的表達低于對照組(P0.05)。染毒72h后,與對照組比較,各染鉛組細胞ERK2及JNK1蛋白的表達均降低(P0.05);500μM染鉛組細胞ERK1的表達低于對照組(P0.05);100μM染鉛組細胞JNK2的表達高于對照組(P0.05),500μM染鉛組細胞JNK2的表達低于對照組(P0.05);JNK3蛋白在100μM及500μM染鉛組細胞中的表達均低于對照組(P0.05);100μM及500μM染鉛組細胞P38的表達低于對照組(P0.05)。6.醋酸鉛對PC12細胞APP、INSR、AKT、IDE和IGF1/IGF1R轉錄水平的影響PCR結果顯示,醋酸鉛處理12h后,APP m RNA在各染鉛組細胞中的表達無明顯改變(P0.05);20μM染鉛組細胞INSR m RNA的表達低于對照組(P0.05);各染鉛組細胞AKT m RNA的表達均低于對照組(P0.05);各實驗組細胞IDE m RNA的表達無顯著差異(P0.05);20μM和100μM染鉛組細胞IGF1/IGF1R m RNA的表達均低于對照組(P0.05)。染毒24h后,20μM及100μM染鉛組細胞APP m RNA的表達高于對照組(P0.05);100μM染鉛組細胞INSR m RNA的表達低于對照組(P0.05);20μM及100μM染鉛組細胞AKT m RNA的表達低于對照組(P0.05);20μM染鉛組細胞IDE m RNA的表達低于對照組(P0.05);100μM染鉛組細胞IGF1 m RNA的表達低于對照組(P0.05),各實驗組細胞IGF1R m RNA的表達無明顯差異(P0.05)。染毒72h后,20μM及100μM染鉛組細胞APP m RNA的表達均低于對照組(P0.05);僅20μM染鉛組細胞INSR m RNA的表達低于對照組(P0.05);AKT m RNA在20μM及100μM染鉛組細胞中的表達低于對照組(P0.05);IDE m RNA在各實驗組中的表達無明顯改變(P0.05);20μM和100μM醋酸染鉛組細胞IGF1/IGF1R m RNA的表達均低于對照組(P0.05)。結論1.孕哺期母體鉛暴露可導致仔鼠血鉛和海馬鉛濃度明顯升高,抑制Aβ降解酶IDE及神經生長因子NGF的表達,損傷仔鼠的空間學習能力,該損傷效應呈暴露濃度依賴性。提示子代學習記憶能力障礙、腦AD樣神經退行性變與孕哺期母體鉛暴露密切相關。2.細胞染毒實驗結果與動物研究結果相一致。醋酸鉛暴露會對PC12細胞AD相關蛋白表達產生影響,該過程可能涉及胞內信號通路蛋白的異常表達。鉛暴露在抑制ERK2、JNK1/3及P38蛋白表達的同時,刺激了ERK1和JNK2 MAPKs的表達。說明鉛暴露可通過誘發(fā)信號通路表達異常來誘導AD樣病變的產生。
[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
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R749.16
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本文編號：2210568