本文選題：阿爾茨海默病　切入點：東莨菪堿　出處：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:阿爾茨海默病(Alzheimer disease,AD)是最常見的老年期癡呆類型,其臨床特征表現(xiàn)為隱襲起病,進行性的記憶和認知功能減退,多伴有人格、精神異常,是年齡相關的神經(jīng)系統(tǒng)退行性疾病;病理學特征表現(xiàn)為β-淀粉樣蛋白(β-amyloid,Aβ)沉積于神經(jīng)元外形成大量老年斑(Senile plaques,SP)和神經(jīng)元內(nèi)形成以過度磷酸化的微管相關蛋白tau為主要成分的神經(jīng)原纖維纏結(Neurofibrillary tangles,NFTs),同時伴有膽堿能神經(jīng)元數(shù)量、突觸數(shù)量的明顯減少。迄今為止,AD的發(fā)病機制仍不是十分明確,當前國內(nèi)外醫(yī)學界主要有以下幾種學說:膽堿能神經(jīng)損傷學說、線粒體損傷學說、氧化應激學說、遺傳與基因突變學說等,但目前無一學說能闡明其發(fā)病機制。東莨菪堿(scopolamine,SCO)是M型膽堿受體拮抗劑,可阻斷大腦皮層和海馬區(qū)乙酰膽堿對M受體的激動作用,從而對記憶獲得產(chǎn)生阻抑效應,誘導癡呆,已經(jīng)被廣泛應用在神經(jīng)營養(yǎng)藥物的評估上。腹腔給藥3mg/Kg后可造成動物記憶功能障礙。東莨菪堿用于誘導動物記憶障礙模型具有簡單、經(jīng)濟、方便等優(yōu)點,故常應用于抗AD活性化合物的初步篩選。茴拉西坦(aniracetam)作為γ-氨基丁酸的環(huán)化衍生物,為腦代謝增強藥,對于改善抑郁癥患者的癥狀方面具有長期效果,同時,茴拉西坦還有抗焦慮的作用。其主要作用機制可能是通過調節(jié)谷氨酸受體中的AMPA受體和類乙酰膽堿能系統(tǒng),但國內(nèi)尚未見將茴拉西坦應用東莨菪堿致癡呆模型中,本實驗以東莨菪堿致癡呆作為AD的模型,探討AMPA受體(Glu R1)、突觸可塑性蛋白的表達情況,并探討茴拉西坦是否可以通過激活谷氨酸受體磷酸化和增高突觸蛋白的表達來改善AD小鼠的認知功能。方法:SPF級雄性BALB/c小鼠96只,全部動物購自北京華維通利華實驗動物技術有限公司,飼養(yǎng)于河北省人民醫(yī)院臨床研究中心動物室內(nèi)。由動物中心的飼養(yǎng)員管理,以標準的鼠糧及純凈的飲水飼養(yǎng)。將小鼠隨機分為6組:vehicle組(生理鹽水3mg/kg,vehicle group)、模型組(SCO 3mg/kg,SCO group)、低劑量組(SCO 3mg/kg+茴拉西坦40mg/kg,L-A group)、中劑量組(SCO 3mg/kg+茴拉西坦80mg/kg,M-A group)、高劑量組(SCO3mg/kg+茴拉西坦160mg/kg,H-A group)、石杉堿甲組(SCO 3mg/kg+石杉堿甲0.15mg/kg,Huper-A group),每組16只,SCO皮下注射日一次,連續(xù)給藥60天,茴拉西坦和石杉堿甲在第50天灌胃給藥,均為日一次,連續(xù)給藥10天。造模完成后進行新物體識別實驗,實驗完成取小鼠海馬組織,采用Western blot蛋白分析法測定ERK1/2、p-ERK1/2、Glu R1、p-Glu R1Ser845、PSD-95、SYN在海馬組織蛋白表達量;采用RT-q PCR技術測定ERK1/2、Glu R1、PSD-95、SYN的m RNA表達量。采用免疫組化方法測定海馬組織PSD-95、SYN的陽性細胞數(shù)。采用專用試劑盒檢測乙酰膽堿酯酶活性。結果:1茴拉西坦改善東莨菪堿引起的小鼠認知功能下降與vehicle組相比,SCO組小鼠的認知指數(shù)明顯降低(P0.01),這說明東莨菪堿明顯降低了小鼠的認知功能,造模成功。而經(jīng)茴拉西坦治療后,與SCO組相比,M-A和H-A組小鼠認知指數(shù)均顯著升高(P0.01),說明茴拉西坦可以改善小鼠的認知功能。2茴拉西坦對突觸可塑性相關蛋白的影響2.1茴拉西坦激活東莨菪堿小鼠海馬ERK1/2蛋白表達磷酸化6組之間的ERK1/2蛋白表達水平?jīng)]有明顯統(tǒng)計學差異(P0.05);與vehicle組相比,SCO組小鼠海馬組織p-ERK1/2蛋白表達明顯下降(P0.01),說明造模成功;而經(jīng)茴拉西坦治療后,與SCO組相比,M-A和H-A組海馬p-ERK1/2蛋白表達水平顯著升高(P0.01);陽性對照組蛋白水平p-ERK1/2表達明顯升高(P0.01)。2.2茴拉西坦激活東莨菪堿小鼠海馬Glu R1蛋白表達磷酸化6組之間的Glu R1蛋白表達水平?jīng)]有明顯統(tǒng)計學差異(P0.05);與vehicle組相比,SCO組小鼠海馬組織p-Glu R1蛋白表達明顯下降(P0.01),說明造模成功;而經(jīng)茴拉西坦治療后,與SCO組相比,H-A組海馬p-Glu R1蛋白表達水平顯著升高(P0.01);陽性對照組蛋白水平p-Glu R1表達明顯升高(P0.01)。2.3茴拉西坦減輕東莨菪堿小鼠海馬突觸蛋白PSD-95、SYN表達的改變與vehicle組相比,SCO組小鼠海馬組織PSD-95、SYN蛋白表達明顯下降(P0.01,P0.01);而經(jīng)茴拉西坦治療后,與SCO組相比,L-A組海馬PSD-95、SYN蛋白水平均顯著升高(P0.01,P0.01);陽性對照組PSD-95、SYN蛋白水平表達明顯升高(P0.01,P0.01)。免疫組織化學染色后可見蛋白表達水平的變化主要是在海馬CA1區(qū)。3茴拉西坦上調東莨菪堿小鼠海馬ERK1/2、Glu R1、SYN、PSD-95基因的表達與vehicle組相比,SCO組小鼠海馬組織ERK1/2、Glu R1、SYN、PSD-95基因表達均明顯下降(P0.01),而經(jīng)茴拉西坦治療后,與SCO組相比,M-A和H-A組海馬ERK1/2基因水平均顯著升高(P0.05,P0.01);與SCO組相比,H-A組海馬Glu R1基因水平均顯著升高(P0.01);與SCO組相比,L-A組海馬PSD-95、SYN基因水平均即顯著升高(P0.01、P0.01),陽性對照組基因水平相應基因表達均明顯升高(P0.01)。4乙酰膽堿酯酶活性測定結果與vehicle組相比,石杉堿甲組乙酰膽堿酯酶活性明顯下降(P0.01),而SCO組乙酰膽堿酯酶活性與vehicle組相比差異沒有統(tǒng)計學意義(P0.05),同樣,茴拉西坦治療組對于乙酰膽堿酯酶活性的影響也沒有統(tǒng)計學意義(P0.05)。結論:1茴拉西坦可以改善東莨菪堿引起的小鼠學習記憶能力下降。2茴拉西坦可以通過增加谷氨酸受體的磷酸化和突觸相關蛋白的表達來提高東莨菪堿小鼠的學習記憶能力。
[Abstract]:Objective: Alzheimer's disease (Alzheimer disease AD) is the most common type of senile dementia, the clinical features of insidious onset, progressive memory and cognitive dysfunction, accompanied by personality, mental disorders, is a neurodegenerative disease related to age; pathological features of beta amyloid protein (beta -amyloid and A beta) deposition in neurons formed outside a large number of senile plaques (Senile plaques, SP) and neuronal neurofibrillary tangle formation by hyperphosphorylation of microtubule associated protein tau as the main component (Neurofibrillary tangles, NFTs), accompanied by the number of cholinergic neurons, significantly reduced the number of synapses so far., the pathogenesis of AD is not very clear, the medical profession at home and abroad are mainly the following: the theory of cholinergic nerve damage theory, mitochondrial damage theory, oxidative stress theory, genetics and gene mutation The theory, but there is no theory can explain its pathogenesis. Scopolamine (scopolamine, SCO) is a type M cholinergic receptor antagonist, blocked acetylcholine in the cerebral cortex and hippocampus of M receptor agonist, which have inhibitory effect on memory access, induced dementia, has been widely used in the evaluation of neurotrophic drug. After intraperitoneal Administration of 3mg/Kg can cause memory impairment induced by scopolamine in animal. The animal model of memory disorder is simple, economic, convenient, preliminary screening is often used in anti AD active compounds. Lacita fennel (aniracetam) as ring derivatives of gamma aminobutyric acid, brain metabolism drugs, has long the effect in improving the symptoms of depression at the same time, Ra Laci Staw and anti anxiety. The main mechanism may be through the regulation of glutamate receptor in AMPA receptor And the cholinergic system, but there is no dementia model induced by scopolamine in the application of Ra Laci Staw, in this experiment, scopolamine induced dementia as the AD model, to investigate the AMPA receptor (Glu R1), the expression of synaptic plasticity protein, and to explore the cognitive function of Ra Laci Staw whether by expression of activation of glutamate receptor phosphorylation and increased synaptic to improve the AD protein in mice. Methods: SPF male BALB/c 96 mice all animal was purchased from Beijing Hua Lihua Experimental Animal Technology Co. Ltd., raised in Hebei People's Hospital clinical research laboratory animal research center. By the breeder management of Animal Center, feeding with the standard rat food and clean drinking water. The mice were randomly divided into into 6 groups: group vehicle (normal saline 3mg/kg, vehicle group), model group (SCO 3mg/kg, SCO group), low dose group (SCO 3mg/kg+ 40mg/kg L-A group La zetham fennel,), in Dose group (SCO 3mg/kg+ 80mg/kg M-A group aniracetam, high dose group (SCO3mg/kg+), aniracetam 160mg/kg, H-A group), huperzine a group (SCO 3mg/kg+ huperzine 0.15mg/kg, Huper-A group), 16 rats in each group, SCO subcutaneous injection once a day, continuous administration for 60 days, fennel Tracy Tanzania and huperzine A in fiftieth days were administered orally, once a day, continuous administration for 10 days. After modeling the new object recognition experiment, experiment the mice hippocampus, determination of ERK1/2, analysis by using the method of Western blot protein p-ERK1/2, Glu R1, p-Glu R1Ser845, PSD-95, expression SYN in hippocampus protein; Determination of ERK1/2, using RT-q PCR technology Glu R1, PSD-95, RNA SYN. The expression of M was measured by immunohistochemical method of PSD-95 in the hippocampus, the number of SYN positive cells. Acetylcholinesterase activity was measured by special kit. Results: 1 aniracetam scopolamine induced small In cognitive decline compared with the vehicle group, cognitive index in group SCO were significantly lower (P0.01), indicating that scopolamine significantly reduces the cognitive function in mice, animal model. By Lacita fennel after treatment, compared with group SCO, M-A and H-A group were significantly elevated both cognitive index (P0.01), description effect of aniracetam can improve the cognitive function of.2 mice Lacita fennel proteins related to synaptic plasticity 2.1 aniracetam activation of scopolamine in mice ERK1/2 protein expression in hippocampus phosphorylation between the 6 groups in the expression level of ERK1/2 protein had no obvious statistical difference (P0.05); compared with vehicle group, the expression of SCO p-ERK1/2 protein in the hippocampus of mice significantly decreased (P0.01), indicating that the model was successful; by aniracetam after treatment, compared with the SCO group, the expression level of M-A and p-ERK1/2 protein in hippocampus of H-A group increased significantly (P0.01); the protein level of p-ERK1/ in the positive control group 2 expression was significantly increased (P0.01).2.2 aniracetam activation of scopolamine in mice hippocampal Glu protein expression of R1 phosphorylation of Glu between the 6 groups R1 protein expression level showed no statistically significant differences (P0.05); compared with vehicle group, SCO p-Glu expression of R1 protein in hippocampus of mice decreased significantly (P0.01), indicating that the model was successful; and by aniracetam after treatment, compared with the SCO group, the expression level of H-A in group p-Glu significantly increased R1 protein (P0.01) positive control group; p-Glu protein level significantly increased the expression of R1 (P0.01).2.3 aniracetam reduce mouse hippocampal synaptic protein PSD-95 induced by scopolamine, the change of SYN expression compared with vehicle group, hippocampus SCO mice in group PSD-95, SYN protein expression was significantly decreased (P0.01, P0.01); and by aniracetam after treatment, compared with the SCO group, L-A group, PSD-95 in hippocampal SYN protein levels were significantly increased (P0.01, P0.01); the positive control group PSD-95, SYN protein The level of expression was significantly increased (P0.01, P0.01). Immunohistochemical staining showed protein expression level changes mainly in the hippocampal CA1 region.3 upregulation of scopolamine in mice hippocampus ERK1/2 Hui Lacita, Glu R1, SYN, PSD-95 gene expression compared with vehicle group, SCO group of mice sea horse tissue ERK1/2, Glu R1, SYN, PSD-95 gene expression decreased significantly (P0.01), and by aniracetam after treatment, compared with the SCO group, M-A group and H-A ERK1/2 gene in the hippocampus were significantly increased (P0.05, P0.01); compared with SCO group, the level of H-A group in hippocampus Glu of R1 gene was significantly elevated (P0.01); compared with the SCO group. In group L-A, PSD-95, SYN gene levels were increased significantly (P0.01, P0.01), the corresponding gene expression level of gene positive control group were significantly higher (P0.01).4 determination of acetylcholinesterase activity compared with vehicle group, huperzine a group of acetylcholinesterase activity decreased obviously (P0.0 1), and SCO group acetylcholinesterase activity compared with vehicle group, the difference was not statistically significant (P0.05). Similarly, Ra Laci Staw treatment group effect on acetylcholinesterase activity was also not statistically significant (P0.05). Conclusion: 1 aniracetam induced by scopolamine can improve the learning and memory ability of mice decreased.2 aniracetam can improve learning and memory the ability of scopolamine in mice by increasing the expression of synapse associated protein phosphorylation and glutamate receptors.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.16

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