本文選題：阿爾茨海默病 + 阿司匹林��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：阿爾茨海默病(Alzheimer's disease,AD)是一種多病因、發(fā)病機(jī)制尚不完全清楚的神經(jīng)退行性疾病,以進(jìn)行性認(rèn)知功能下降、學(xué)習(xí)記憶能力減退、神經(jīng)精神病學(xué)癥狀和日常行為異常等為主要臨床特征,而由腦神經(jīng)細(xì)胞外異常沉積的淀粉樣β蛋白(Amyloid-βprotein,Aβ)和細(xì)胞內(nèi)tau蛋白高度磷酸化聚集形成的神經(jīng)纖維纏結(jié)(neurofibrillary tangles,NFTs)誘發(fā)的神經(jīng)炎性反應(yīng)、神經(jīng)元凋亡、Aβ的生成與清除失衡為其主要病理學(xué)特征。AD是全球老年癡呆患者中最常見(jiàn)的類型,隨著全球人口老齡化的加劇,其發(fā)病率也將快速增加,這將給患者、家人和社會(huì)帶來(lái)沉重的經(jīng)濟(jì)負(fù)擔(dān)。雖然對(duì)AD發(fā)病機(jī)制的研究越來(lái)越深入,但AD在當(dāng)前仍然是一種不可治愈且難以阻止其進(jìn)程的疾病,研發(fā)治療AD的有效藥物也成為目前面臨的一個(gè)巨大挑戰(zhàn)。大量研究發(fā)現(xiàn),非甾體類抗炎藥(non-steroid anti-inflammatory drugs,NSAIDs)在防治AD中具有潛在的有益作用。所以,針對(duì)AD的發(fā)病機(jī)制,如能在現(xiàn)有藥物基礎(chǔ)上,探索一種經(jīng)濟(jì)有效的防治藥物將會(huì)產(chǎn)生巨大的社會(huì)意義和經(jīng)濟(jì)效益。大量研究證實(shí)中樞神經(jīng)炎性反應(yīng)在AD的發(fā)生和進(jìn)展過(guò)程中發(fā)揮著關(guān)鍵作用。神經(jīng)炎性反應(yīng)可引起促炎性細(xì)胞因子白細(xì)胞介素1β(interleukin 1β,IL-1β)、腫瘤壞死因子α(tumor necrosis factor,TNF-α)等生成增加,而抗炎因子白細(xì)胞介素4(interleukin4,IL-4)、白細(xì)胞介素10(interleukin 10,IL-10)等不足也可能促進(jìn)了神經(jīng)炎性反應(yīng)。IL-1β和TNF-α的神經(jīng)毒性不僅可以損傷神經(jīng)元和神經(jīng)突觸,反過(guò)來(lái)又激活NF-κB,活化的核轉(zhuǎn)錄因子κB(nuclear transcription factor,NF-κB)又可以誘導(dǎo)IL-1β、TNF-α、誘導(dǎo)型一氧化氮合酶(induced nitric oxide synthase,i NOS)和脂多糖(Lipopolysaccharide,LPS)等細(xì)胞因子表達(dá),形成一個(gè)慢性神經(jīng)炎性反應(yīng)的惡性回路。隨著神經(jīng)炎性反應(yīng)的持續(xù)存在,抗炎反應(yīng)對(duì)促炎刺激持續(xù)缺乏,致使抗炎/促炎因子失衡,進(jìn)一步促進(jìn)神經(jīng)炎性反應(yīng)的發(fā)展。因此,通過(guò)抗炎藥物抑制神經(jīng)炎性反應(yīng)、保護(hù)神經(jīng)元,進(jìn)而改善神經(jīng)病理學(xué)變化可能是一種潛在的防治AD的新策略。阿司匹林為經(jīng)典的NSAIDs,大量臨床研究認(rèn)為長(zhǎng)期服用阿司匹林(Aspirin,Asp)的人群AD發(fā)病率低于未服用人群,阿司匹林在AD防治方面具有潛在可能性。因此,本研究通過(guò)對(duì)大鼠進(jìn)行側(cè)腦室注射淀粉樣β蛋白25-35(Amyloid-βprotein 25-35,Aβ25-35)建立AD動(dòng)物模型,探究阿司匹林的抗炎作用對(duì)AD模型大鼠空間學(xué)習(xí)能力及海馬組織中促炎/抗炎因子IL-1β、TNF-α、IL-4和IL-10以及NF-κB、i NOS表達(dá)水平的影響,以期能為阿司匹林在防治AD中開(kāi)展臨床試驗(yàn)研究和實(shí)際應(yīng)用提供部分實(shí)驗(yàn)室依據(jù)。研究目的中樞神經(jīng)系統(tǒng)(central nervous system,CNS)的炎性反應(yīng)和抗炎/促炎因子表達(dá)失衡被認(rèn)為在AD的發(fā)生發(fā)展進(jìn)程中發(fā)揮了關(guān)鍵作用。本課題通過(guò)Asp干預(yù)Aβ25-35誘導(dǎo)的AD模型鼠,研究阿司匹林對(duì)AD模型大鼠空間學(xué)習(xí)記憶能力、海馬區(qū)炎性因子(IL-1β、TNF-α、IL-4和IL-10)表達(dá)水平以及抗炎/促炎因子微平衡的影響,并進(jìn)一步探討阿司匹林對(duì)AD的潛在防治作用及可能機(jī)制。研究方法40只雄性SD大鼠按照完全隨機(jī)的方法分為四個(gè)實(shí)驗(yàn)組,10只/組。(1)對(duì)照組:自由飲用蒸餾水,喂養(yǎng)3周后,使用腦立體定向儀進(jìn)行大鼠右側(cè)側(cè)腦室注射5μl的無(wú)菌生理鹽水,再給予蒸餾水喂養(yǎng)3周;自由進(jìn)食。(2)AD模型組:自由飲用蒸餾水,喂養(yǎng)3周后,使用腦立體定向儀進(jìn)行大鼠右側(cè)側(cè)腦室注射5μl的Aβ25-35(10mmol/L)溶液,再給予蒸餾水喂養(yǎng)3周;自由進(jìn)食。(3)低劑量阿司匹林干預(yù)組(1mg/ml):自由飲用蒸餾水,喂養(yǎng)3周后,使用腦立體定向儀進(jìn)行大鼠右側(cè)側(cè)腦室注射5μl的Aβ25-35(10mmol/L)溶液,再給予蒸餾水喂養(yǎng)3周;自由進(jìn)食。(4)高劑量阿司匹林干預(yù)組(2mg/ml):自由飲用蒸餾水,喂養(yǎng)3周后,使用腦立體定向儀進(jìn)行大鼠右側(cè)側(cè)腦室注射5μl的Aβ25-35(10mmol/L)溶液,再給予蒸餾水喂養(yǎng)3周;自由進(jìn)食。側(cè)腦室注射術(shù)后繼續(xù)喂養(yǎng)3周后,通過(guò)Morris水迷宮檢測(cè)大鼠空間學(xué)習(xí)、記憶能力;采用雙抗體夾心ELISA法檢測(cè)海馬區(qū)組織中IL-1β、TNF-α、IL-4和IL-10的表達(dá)水平;海馬區(qū)組織制作病理切片,免疫組化染色觀察NF-κB和i NOS表達(dá)情況,尼氏染色觀察神經(jīng)元凋亡情況。研究結(jié)果(1)空間學(xué)習(xí)記憶能力:與對(duì)照組比較,AD模型大鼠空間學(xué)習(xí)記憶能力顯著下降(P0.001),1mg/ml Asp組大鼠與對(duì)照組差異有統(tǒng)計(jì)學(xué)意義(P0.05),2mg/ml Asp組大鼠與對(duì)照組比較無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);與AD模型組比較,1mg/ml Asp組大鼠與模型組差異有統(tǒng)計(jì)學(xué)意義(P0.05),2mg/ml Asp組大鼠與模型組差異有統(tǒng)計(jì)學(xué)意義(P0.01);Asp干預(yù)組組間比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(2)海馬區(qū)IL-1β表達(dá):與對(duì)照組比較,AD模型組大鼠海馬組織中IL-1β表達(dá)水平升高顯著(P0.001),Asp干預(yù)組與對(duì)照組差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);與AD模型組比較,低、高劑量Asp干預(yù)組IL-1β表達(dá)水平均明顯下降,差異均有統(tǒng)計(jì)學(xué)意義(P0.01);Asp干預(yù)組組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(3)海馬區(qū)TNF-α表達(dá):與對(duì)照組比較,模型組大鼠海馬組織中TNF-α表達(dá)水平顯著升高(P0.001),1mg/ml Asp組與對(duì)照組差異有統(tǒng)計(jì)學(xué)意義(P0.01),2mg/ml Asp組與對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);Asp干預(yù)組與AD模型組比較,2mg/ml Asp組大鼠海馬組織中TNF-α表達(dá)水平明顯降低(P0.01),而1mg/ml Asp組與AD模型組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);Asp干預(yù)組組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(4)海馬區(qū)IL-4表達(dá):與對(duì)照組大鼠比較,AD模型組大鼠海馬組織中IL-4表達(dá)水平下降顯著(P0.001),1mg/ml Asp組與對(duì)照組差異有統(tǒng)計(jì)學(xué)意義(P0.01),2mg/ml Asp組與對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);與AD模型組比較,2mg/ml Asp組大鼠海馬區(qū)組織中IL-4表達(dá)水平明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.01),1mg/ml Asp組與AD模型組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);Asp干預(yù)組組間比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(5)海馬區(qū)IL-10表達(dá):與對(duì)照組比較,AD模型組大鼠海馬區(qū)組織中IL-10表達(dá)水平下降顯著(P0.01),1mg/ml Asp組與對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),2mg/ml Asp組與對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);與模型組比較,2mg/ml Asp組大鼠海馬區(qū)組織中IL-10表達(dá)水平明顯升高(P0.05),1mg/ml Asp組大鼠與AD模型組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);Asp干預(yù)組組間比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(6)海馬區(qū)NF-κB和i NOS陽(yáng)性表達(dá):與對(duì)照組比較,AD模型組海馬區(qū)組織中NF-κB和i NOS陽(yáng)性表達(dá)明顯顯著增加,神經(jīng)元缺失明顯,而Asp干預(yù)組海馬區(qū)組織中均有不同程度的NF-κB、i NOS陽(yáng)性表達(dá)和神經(jīng)元缺失,其中2mg/ml Asp干預(yù)組與對(duì)照組大致相當(dāng)。(7)海馬區(qū)神經(jīng)元:與對(duì)照組比較,AD模型組海馬區(qū)神經(jīng)元缺失明顯,而Asp干預(yù)組海馬區(qū)均有不同程度神經(jīng)元缺失,其中2mg/ml Asp干預(yù)組神經(jīng)元缺失不明顯,與對(duì)照組大致相當(dāng)。研究結(jié)論阿司匹林干預(yù)可保護(hù)AD模型大鼠空間學(xué)習(xí)記憶能力;阿司匹林通過(guò)抑制炎性反應(yīng)、NF-κB通路活化及i NOS表達(dá),下調(diào)促炎因子IL-1β、TNF-α水平和上調(diào)抗炎因子IL-4、IL-10水平,進(jìn)而調(diào)整促炎/抗炎因子失衡狀態(tài),在AD發(fā)病過(guò)程中抑制神經(jīng)炎性反應(yīng)發(fā)展,發(fā)揮抗炎、保護(hù)神經(jīng)元和學(xué)習(xí)記憶能力的作用。
[Abstract]:Alzheimer's disease (AD) is a multiple cause of neurodegenerative disease whose pathogenesis is not completely clear. The main clinical features are progressive cognitive impairment, impairment of learning and memory, neuropsychiatry symptoms and daily behavior abnormalities, and amyloid beta protein (amyloid beta protein), which is abnormally deposited outside the nerve cells of the brain. Amyloid- beta protein, A beta, and highly phosphorylated intracellular tau protein aggregation (neurofibrillary tangles, NFTs) induced neuroinflammatory response, neuronal apoptosis, the formation and clearance of A beta, the main pathological feature of A beta, as the most common type of age idiot in the world, with the aging of the global population. The increasing incidence of the disease will also increase rapidly, which will bring a heavy financial burden to patients, family and society. Although the research on the pathogenesis of AD is becoming more and more deep, AD is still a disease that is not curable and difficult to prevent its process. The development of effective drugs for the treatment of AD has also become a huge challenge. Quantitative studies have found that non-steroid anti-inflammatory drugs (NSAIDs) has a potential beneficial effect in the prevention and control of AD. Therefore, in view of the pathogenesis of AD, the exploration of an economic and effective control drug on the basis of existing drugs will produce great social and economic benefits. A large number of studies have confirmed the center. The neuroinflammatory response plays a key role in the development and progression of AD. The neuroinflammatory response can cause the increase of pro-inflammatory cytokines interleukin 1 beta (interleukin 1 beta, IL-1 beta), tumor necrosis factor alpha (tumor necrosis factor, TNF- alpha), and the anti inflammatory factor interleukin 4 (interleukin4, IL-4), and interleukin 10 (I). Nterleukin 10, IL-10) and other deficiencies may also promote the neuroinflammatory response to the neurotoxicity of.IL-1 beta and TNF- alpha, which not only damage neurons and synapses, but also activate NF- kappa B, and the activated nuclear factor kappa B (nuclear transcription factor, NF- kappa B) can also induce the beta, alpha, inducible nitric oxide synthase (inducible nitric oxide synthase). The expression of cytokines such as oxide synthase, I NOS) and lipopolysaccharide (Lipopolysaccharide, LPS) forms a malignant loop of chronic inflammatory response. With the persistent neuroinflammatory response, the anti-inflammatory response to proinflammatory stimuli is continuously lacking, causing anti inflammatory / proinflammatory factors to balance and further promoting the development of neuroinflammatory response. Anti inflammatory drugs to inhibit neuroinflammatory response, protect neurons, and then improve neuropathological changes may be a potential new strategy for the prevention and control of AD. Aspirin is a classic NSAIDs. A large number of clinical studies believe that the incidence of AD in people who have long taken aspirin (Aspirin, Asp) is lower than that of non taking people and aspirin in the prevention and treatment of AD. Therefore, in this study, the rat model of AD was established by injecting amyloid beta protein 25-35 (Amyloid- beta protein 25-35, A beta 25-35) into the lateral ventricle of the rat, to explore the spatial learning ability of aspirin in AD model rats and the proinflammatory / anti-inflammatory factor IL-1 beta, TNF- a, IL-4 and IL-10, and NF- kappa B in the hippocampus. The effect of S expression level is expected to provide some laboratory basis for the clinical trials and practical applications of aspirin in the prevention and control of AD. The inflammatory response of the central nervous system (CNS) and the imbalance of the expression of anti-inflammatory / proinflammatory factors are considered to play a key role in the development of AD. The AD model rats induced by A beta 25-35 were used to study the spatial learning and memory ability of aspirin on AD model rats, the expression level of inflammatory factors (IL-1 beta, TNF- a, IL-4 and IL-10) in the hippocampus and the effect of the micro balance of anti-inflammatory and proinflammatory factors in the hippocampus, and further explore the potential preventive and therapeutic effects of aspirin on AD and its possible mechanism. 40 male SD rats were divided into four experimental groups according to the complete random method, 10 / group. (1) the control group: free drinking distilled water. After feeding for 3 weeks, the right side ventricle of the rat was injected with the sterile saline of 5 mu in the right lateral ventricle of the rat and fed with distilled water for 3 weeks. (2) the free drinking of distilled water and feeding for 3 weeks (2) AD model group. After that, the right lateral ventricle of the rat was injected with 5 L A beta 25-35 (10mmol/L) solution, and then fed with distilled water for 3 weeks, and free feeding. (3) low dose aspirin intervention group (1mg/ml): free drinking distilled water, and after feeding for 3 weeks, the right lateral ventricle of rats was injected with 5 L A beta 25-35 (10mmol/L) by injection of brain stereotaxic apparatus. The liquid was given to distilled water for 3 weeks; free feeding. (4) high dose aspirin intervention group (2mg/ml): free drinking distilled water. After feeding for 3 weeks, the right lateral ventricle of the rat was injected with 5 u l A beta 25-35 (10mmol/L), and then fed with distilled water for 3 weeks. Morris water maze was used to detect the spatial learning and memory ability of rats, and the expression level of IL-1 beta, TNF- a, IL-4 and IL-10 in hippocampus tissue was detected by double antibody sandwich ELISA; the expression of NF- kappa B and I NOS was observed by immunohistochemical staining in hippocampus tissue, and neuronal apoptosis was observed by immunohistochemical staining, and the results of Nissl staining were observed. (1) space Learning and memory ability: compared with the control group, the spatial learning and memory ability of the AD model rats decreased significantly (P0.001). The difference between the 1mg/ml Asp group and the control group was statistically significant (P0.05), and there was no significant difference between the 2mg/ml Asp group and the control group (P0.05), and the difference between the 1mg/ml Asp group and the model group was statistically significant compared with the AD model group. (P0.05) the difference between the 2mg/ml Asp group and the model group was statistically significant (P0.01), and the difference between the Asp intervention group was not statistically significant (P0.05). (2) the expression of IL-1 beta in the hippocampus: the increase of IL-1 beta expression in the hippocampus of the AD model group was significantly higher than that in the control group (P0.001), and there was no significant difference between the Asp intervention group and the control group (P0.0). 5): compared with the AD model group, the expression level of IL-1 beta in the low and high dose Asp intervention group was significantly decreased, the difference was statistically significant (P0.01), and there was no statistical difference between the Asp intervention group (P0.05). (3) the expression of TNF- a in the hippocampus: compared with the control group, the expression of TNF- a in the hippocampus of the model rats was significantly increased (P0.001), 1mg/ml Asp. There was significant difference between the group and the control group (P0.01), but there was no significant difference between the 2mg/ml Asp group and the control group (P0.05), and the expression level of TNF- alpha in the hippocampus of 2mg/ml Asp group was significantly lower than that of the AD model group (P0.01), but there was no significant difference between the 1mg/ml Asp group and the model group. There was no statistical significance (P0.05). (4) the expression of IL-4 in the hippocampus: compared with the control group, the level of IL-4 expression in the hippocampus of the AD model rats decreased significantly (P0.001), and the difference between the 1mg/ml Asp group and the control group was statistically significant (P0.01), and the 2mg/ml Asp group was different from the control group (P0.05); compared with the AD model group, the group was larger than the AD model group. The expression level of IL-4 in the rat hippocampus was significantly increased (P0.01), and there was no significant difference between the 1mg/ml Asp group and the AD model group (P0.05), and there was no statistical difference between the Asp intervention group and the Asp intervention group (P0.05). (5) the IL-10 expression in the hippocampus: the IL-10 expression level in the hippocampus of the AD model group was lower than that in the control group. There was no significant difference between the 1mg/ml Asp group and the control group (P0.05), but there was no significant difference between the 2mg/ml Asp group and the control group (P0.05). Compared with the model group, the IL-10 expression level of the hippocampus tissue in the group 2mg/ml Asp group was significantly higher (P0.05). There was no statistical difference between the rats of the 2mg/ml Asp group and the P0.01 group. There was no significant difference between the groups (P0.05). (6) the positive expression of NF- kappa B and I NOS in the hippocampus: compared with the control group, the positive expression of NF- kappa B and I NOS increased significantly in the hippocampus tissue of the AD model group, and the neuron deletion was obvious. 2mg/ml Asp intervention group was roughly equivalent to the control group. (7) hippocampal neurons: compared with the control group, the neuron loss in the hippocampus of the AD model group was obvious, while the hippocampus of the Asp intervention group had different degrees of neuron loss, and the neuron deletion in the 2mg/ml Asp intervention group was not obvious, which was roughly equivalent to the control group. Protecting AD model rats' spatial learning and memory ability; aspirin can inhibit inflammatory response, activation of NF- kappa B pathway and I NOS expression, down-regulation of proinflammatory factor IL-1 beta, TNF- a level and up regulation of IL-4, IL-10 level, and then regulate the state of inflammation and anti-inflammatory factors, inhibit the development of neuroinflammatory reaction in the course of AD hair disease, play anti-inflammatory and protect it. The function of protecting neurons and learning and memory.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16

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