本文選題：鉛　切入點：白藜蘆醇　出處：《鄭州大學》2014年碩士論文

【摘要】：鉛(Lead, Pb)是一種普遍存在的環(huán)境重金屬污染物，有較強的神經(jīng)毒性，并損傷認知功能，發(fā)育期的鉛暴露可以誘發(fā)阿爾茨海默病(Alzheimer’s disease,AD)樣病變的形成。多酚類化合物白藜蘆醇(Resveratrol, Res)對神經(jīng)退行性疾病和急性損傷具有保護作用，Res激活產(chǎn)物沉默信息調(diào)節(jié)因子1(Silent InformationRegulator1, SIRT1)具有抗氧化作用，而氧化應(yīng)激是AD產(chǎn)生的重要機制之一，SIRT1對于氧化應(yīng)激的相關(guān)調(diào)控在鉛致小鼠AD樣病變中可能發(fā)揮作用。 目的 基于鉛致AD淀粉樣變的實驗基礎(chǔ)，以Aβ的形成和認知效應(yīng)為觀察終點，采用Res進行干預(yù)研究，圍繞SIRTl及其效應(yīng)分子FOXO3a、PGC-lα的表達，分析鉛暴露、SIRTl相關(guān)的抗氧化應(yīng)激效應(yīng)與神經(jīng)損傷之間的可能關(guān)系，為AD的病因及發(fā)病機制研究提供理論線索。 方法 1．動物模型建立：選用清潔級C57BL/6健康斷乳（出生后21天）仔鼠35只作為研究對象，由本實驗組進行仔鼠的繁育飼養(yǎng)。按體重隨機分為五組：Res灌胃組、對照組、9M染鉛組、9M染鉛+Res灌胃組、3M染鉛+Res灌胃組。以自由飲用2g/L醋酸鉛溶液的方式進行鉛暴露；與鉛暴露三個月后進行白藜蘆醇灌胃，劑量為50mg/kgBw d，，每周連續(xù)灌胃6天后，停灌1天，持續(xù)六個月。 2．行為學測試：染毒結(jié)束后，進行Morris水迷宮實驗，測定小鼠空間學習記憶能力。 3．組織提取：水迷宮實驗結(jié)束后兩天，進行小鼠麻醉后眼球采血，收集全血及腦組織，分離出皮層，生理鹽水漂洗后，稱重，置于凍存管中-80℃保存。 4．實驗室指標測定：用Z-5000偏振塞曼原子吸收光譜儀測定小鼠血鉛；谷胱甘肽過氧化物酶(GSH-PX)測試試劑盒測定大腦皮層GSH-PX活力；還原型谷胱甘肽測試盒測定小鼠大腦皮層GSH含量；Aβ(1-40)ELISA試劑盒測定小鼠大腦皮層Aβ(1-40)含量；Western blot分析小鼠大腦皮層SIRT1、磷酸化FOXO3a（Ser253）、PGC-lα蛋白表達水平。 結(jié)果 1．Morris水迷宮結(jié)果：前五天小鼠的平均逃避潛伏期隨日期變化逐漸減�。‵=2.799,P0.05）；9M染鉛組、9M染鉛+Res灌胃組的平均逃避潛伏期分別大于對照組、3M染鉛+Res灌胃組（P0.05），9M染鉛組的平均逃避潛伏期大于Res灌胃組（P0.05）。第6天撤去平臺，9M染鉛組和9M染鉛+Res灌胃組的逃避潛伏期分別大于對照組、3M染鉛+Res灌胃組（P0.05）。 2．小鼠血鉛含量：各實驗組小鼠血鉛含量存在差異（F=92.21,P0.001），Res灌胃組、對照組的血鉛含量分別小于9M染鉛組、9M染鉛+Res灌胃組、3M染鉛+Res灌胃組（P0.001）；9M染鉛組、9M染鉛+Res灌胃組的血鉛含量分別大于3M染鉛+Res灌胃組（P0.001）。 3．GSH-Px活力值和GSH含量：各組小鼠大腦皮層GSH含量存在差異（F=6.177，P0.05），Res灌胃組、對照組GSH含量高于9M染鉛+Res灌胃組、9M染鉛組（P0.05），3M染鉛+Res灌胃組GSH含量高于9M染鉛組（P0.05）。 各組小鼠大腦皮層GSH-Px活力值存在差異（F=10.195,P0.001），Res灌胃組GSH-Px活力值大于對照組（P0.05），9M染鉛組、9M染鉛+Res灌胃組、3M染鉛+Res灌胃組GSH-Px活力值小于對照組（P0.05），3M染鉛+Res灌胃組小鼠GSH-Px活力值大于9M染鉛組（P0.05）； 4．Aβ(1-40)含量：各實驗組小鼠大腦皮層Aβ(1-40)含量存在差異（F=3.285，P0.05），9M染鉛組Aβ(1-40)含量分別大于Res灌胃組、對照組、3M染鉛+Res灌胃組（P0.05）。 5．Western blot分析結(jié)果：各實驗組小鼠大腦皮層細胞核內(nèi)SIRT1蛋白表達水平存在差異（F=216.779,P0.001），對照組SIRT1水平高于其他各組（P0.05），Res灌胃組、9M染鉛+Res灌胃組、3M染鉛+Res灌胃組SIRT1水平低于9M染鉛組（P0.05），Res灌胃組SIRT1水平高于3M染鉛+Res灌胃組和9M染鉛+Res灌胃組（P0.05）。 各實驗組小鼠大腦皮層磷酸化FOXO3a表達水平存在差異（F=18.17，P0.001），Res灌胃組磷酸化FOXO3a水平低于其他各組（P0.05），對照組磷酸化FOXO3a水平則高于其他各組（P0.05），9M染鉛組磷酸化FOXO3a水平高于3M染鉛+Res灌胃組（P0.05）。 各實驗組小鼠大腦皮層總蛋白中PGC-lα表達水平存在差異（F=58.632,P0.001），Res灌胃組PGC-lα表達水平高于對照組（P0.05），9M染鉛組、9M染鉛+Res灌胃組、3M染鉛+Res灌胃組PGC-lα表達水平均低于對照組（P0.05），其中3M染鉛+Res灌胃組PGC-lα表達水平高于9M染鉛組；各實驗組小鼠大腦皮層胞漿蛋白中PGC-lα表達水平也存在差異（F=67.327，P0.001），Res灌胃組小鼠PGC-lα表達水平低于對照組（P0.05），9M染鉛組PGC-lα表達水平高于對照組（P0.05），9M染鉛+Res灌胃組、3M染鉛+Res灌胃組PGC-lα表達水平均低于9M染鉛組（P0.05）且與對照組差異無統(tǒng)計意義（P0.05）。 結(jié)論 1.發(fā)育早期鉛暴露可降低小鼠大腦皮層中磷酸化FOXO3a、PGC-lα及大腦皮層細胞核內(nèi)SIRT1表達水平，并增加PGC-lα在胞漿中的滯留，減少GSH-Px的酶活力以及GSH含量，降低抗氧化能力，損傷小鼠空間學習和記憶能力； 2.白藜蘆醇干預(yù)可能通過SIRTI的核質(zhì)轉(zhuǎn)運降低小鼠大腦皮層中的磷酸化FOXO3a表達水平、升高大腦皮層PGC-lα表達水平，增進PGC-lα去乙�；蟮暮藘�(nèi)轉(zhuǎn)運及轉(zhuǎn)錄活性，增加GSH-Px的酶活力及GSH含量，提高抗氧化能力，對鉛致小鼠空間學習記憶能力損傷有保護作用。
[Abstract]:Lead (Lead, Pb) is a ubiquitous environmental heavy metal pollutants, toxicity, and cognitive impairment, developmental lead exposure can induce Alzheimer's disease (Alzheimer 's disease, AD) formed like lesions. The polyphenolic compound resveratrol (Resveratrol, Res) has a protective effect on nerve degenerative diseases and acute injury, activation of Res product of silent information regulator 1 (Silent, InformationRegulator1, SIRT1) with antioxidant effects, and oxidative stress is one of the important mechanisms of AD, SIRT1 in regulation of oxidative stress may play a role in lead induced mouse AD like lesions.
objective
The experimental basis of lead induced AD amyloidosis based on form and cognitive effects of A beta to observe the end point, the intervention study was conducted by Res, around SIRTl and its effector molecule FOXO3a, expression of PGC-l, analysis of lead exposure, the possible relationship between SIRTl anti oxidative stress effects associated with nerve injury, to provide theoretical clues for the study the etiology and pathogenesis of AD.
Method
1. animal model: choose healthy C57BL/6 weaning (postnatal day 21) 35 mice as the research object, the experimental group of rats. Breeding breeding were randomly divided into five groups: Res group, control group, 9M exposure group, 9M lead +Res group 3M, lead +Res gavage group. By drinking 2g/L lead acetate solution of lead exposure; and lead exposure three months after intragastric administration of resveratrol, the dose of 50mg/kgBw D, a weekly intragastric administration for 6 days, stopping perfusion for 1 days, lasted for six months.
2. behavior test: after the end of the drug, the Morris water maze test was carried out to determine the learning and memory ability of the mice.
3. tissue extraction: two days after the water maze test, the blood samples were collected from the eyeballs after anesthesia, and the whole blood and brain tissues were collected. The cortex was isolated. After rinsing, the saline was weighed and stored in the cryopreservation tube at -80 C.
Determination of 4. laboratory index: Z-5000 Polarized Zeeman atomic absorption spectrometer for determination of lead in mice; glutathione peroxidase (GSH-PX) test kit for determination of GSH-PX activity in the cerebral cortex; Determination of GSH content in cerebral cortex glutathione test kit; A beta (1-40) ELISA kit mouse cerebral cortex A beta (1-40) content; Western blot analysis of mouse cerebral cortex SIRT1, phosphorylation of FOXO3a (Ser253), the expression level of PGC-l protein.
Result
The results of the 1.Morris water maze: the average escape latency of five days before the date with mice gradually decreased (F=2.799, P0.05); 9M lead group, the average escape latency of 9M lead +Res gavage group were higher than the control group, 3M lead +Res gavage group (P0.05), the average escape latency of 9M in lead exposed group more than Res group (P0.05). Sixth days removed platform, 9M lead group and 9M lead +Res group the escape latency were higher than the control group, 3M lead +Res gavage group (P0.05).
2. mouse blood lead content: there are differences in each experimental group of mice blood lead levels (F=92.21, P0.001), Res group, blood lead content in control group were less than 9M in lead group, 9M lead +Res gavage group, 3M lead +Res gavage group (P0.001); 9M blood lead group. The lead content of 9M lead +Res gavage group were higher than 3M lead +Res gavage group (P0.001).
3.GSH-Px activity and GSH content: there were differences in GSH content in cerebral cortex of each group (F=6.177, P0.05), Res in gavage group, GSH content in control group was higher than 9M, lead and +Res gavage group, 9M lead exposure group (P0.05), and the level of lead in group B was higher than that in group B (group B).
There were differences in mouse cerebral cortex GSH-Px activity value (F=10.195, P0.001), Res group GSH-Px activity was higher than that of the control group (P0.05), 9M lead group, 9M lead +Res gavage group, 3M lead +Res gavage group GSH-Px activity value is less than the control group (P0.05), 3M +Res was exposed to lead mice gastric GSH-Px activity value greater than 9M in lead group (P0.05);
4.A beta (1-40) content: there was a difference in the content of A beta (1-40) in the cerebral cortex of each experimental group (F=3.285, P0.05), and the content of A beta (1-40) in 9M lead staining group was higher than that in Res gavage group, while in control group, 3M was exposed to lead and +Res in gavage group (P0.05).
5.Western blot analysis: the experimental group of mice cerebral cortex nucleus SIRT1 protein expression level differences (F=216.779, P0.001), the control group SIRT1 was higher than other groups (P0.05), Res group, 9M lead +Res group, 3M lead +Res group SIRT1 level was lower than that of 9M in lead group (P0.05, Res) with SIRT1 level in gastric lead exposed group was higher than that in 3M +Res group and 9M lead +Res gavage group (P0.05).
Differences in the level of expression of each experimental group in cerebral cortex of phosphorylated FOXO3a (F=18.17, P0.001), Res gavage group phosphorylated FOXO3a levels lower than the other groups (P0.05), the control group the level of phosphorylated FOXO3a was higher than other groups (P0.05), 9M lead group FOXO3a phosphorylation level was higher than that of 3M lead +Res gavage group (P0.05).
Differences in the level of PGC-l expression in each experimental group total protein in mouse cerebral cortex (F=58.632, P0.001), Res group PGC-l expression level was higher than that of the control group (P0.05), 9M lead group, 9M lead +Res gavage group, 3M lead +Res group PGC-l expression levels were lower than the control group (P0.05), of which 3M lead +Res group PGC-l expression level was higher than that of 9M in lead group; the experimental group cells of cerebral cortex in mice plasma protein PGC-l expression level differences (F=67.327, P0.001), Res mice fed the PGC-l expression level was lower than that of the control group (P0.05), 9M lead group PGC-l expression level was higher than that of the control group (P0.05), 9M lead +Res gavage group, 3M lead +Res group PGC-l expression levels were lower than that of 9M in lead group (P0.05) and the control group showed no statistical significance (P0.05).
conclusion
1. early developmental lead exposure could decrease the phosphorylation of FOXO3a in mouse cerebral cortex, the expression level of SIRT1 alpha and PGC-l in cerebral cortex of the nucleus, and increased PGC-l alpha stranded in the cytoplasm, decrease of GSH-Px activity and GSH content, decreased antioxidant capacity, impaired spatial learning and memory in mice;
2. resveratrol intervention may reduce the expression level of phosphorylated FOXO3a in the cerebral cortex of mice through nucleocytoplasmic transport of SIRTI, increased cortical PGC-l expression level, to enhance PGC-l alpha nuclear translocation and transcriptional activity after acetylation, increase enzyme activity and GSH content of GSH-Px, improve the antioxidant ability of mice induced by lead the ability of spatial learning and memory has a protective effect on injury.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R285.5

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