本文選題：阿爾茨海默病 + 阿司匹林��； 參考：《第二軍醫(yī)大學》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)病機制尚不完全清楚的神經(jīng)退行性疾病,以進行性認知功能下降、學習記憶能力減退、神經(jīng)精神病學癥狀和日常行為異常等為主要臨床特征,而由腦神經(jīng)細胞外異常沉積的淀粉樣β蛋白(Amyloid-βprotein,Aβ)和細胞內(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、神經(jīng)元凋亡、Aβ的生成與清除失衡為其主要病理學特征。AD是全球老年癡呆患者中最常見的類型,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、家人和社會帶來沉重的經(jīng)濟負擔。雖然對AD發(fā)病機制的研究越來越深入,但AD在當前仍然是一種不可治愈且難以阻止其進程的疾病,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AD的發(fā)病機制,如能在現(xiàn)有藥物基礎上,探索一種經(jīng)濟有效的防治藥物將會產(chǎn)生巨大的社會意義和經(jīng)濟效益。大量研究證實中樞神經(jīng)炎性反應在AD的發(fā)生和進展過程中發(fā)揮著關鍵作用。神經(jīng)炎性反應可引起促炎性細胞因子白細胞介素1β(interleukin 1β,IL-1β)、腫瘤壞死因子α(tumor necrosis factor,TNF-α)等生成增加,而抗炎因子白細胞介素4(interleukin4,IL-4)、白細胞介素10(interleukin 10,IL-10)等不足也可能促進了神經(jīng)炎性反應。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,反過來又激活NF-κB,活化的核轉(zhuǎn)錄因子κB(nuclear transcription factor,NF-κB)又可以誘導IL-1β、TNF-α、誘導型一氧化氮合酶(induced nitric oxide synthase,i NOS)和脂多糖(Lipopolysaccharide,LPS)等細胞因子表達,形成一個慢性神經(jīng)炎性反應的惡性回路。隨著神經(jīng)炎性反應的持續(xù)存在,抗炎反應對促炎刺激持續(xù)缺乏,致使抗炎/促炎因子失衡,進一步促進神經(jīng)炎性反應的發(fā)展。因此,通過抗炎藥物抑制神經(jīng)炎性反應、保護神經(jīng)元,進而改善神經(jīng)病理學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,大量臨床研究認為長期服用阿司匹林(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,本研究通過對大鼠進行側(cè)腦室注射淀粉樣β蛋白25-35(Amyloid-βprotein 25-35,Aβ25-35)建立AD動物模型,探究阿司匹林的抗炎作用對AD模型大鼠空間學習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表達水平的影響,以期能為阿司匹林在防治AD中開展臨床試驗研究和實際應用提供部分實驗室依據(jù)。研究目的中樞神經(jīng)系統(tǒng)(central nervous system,CNS)的炎性反應和抗炎/促炎因子表達失衡被認為在AD的發(fā)生發(fā)展進程中發(fā)揮了關鍵作用。本課題通過Asp干預Aβ25-35誘導的AD模型鼠,研究阿司匹林對AD模型大鼠空間學習記憶能力、海馬區(qū)炎性因子(IL-1β、TNF-α、IL-4和IL-10)表達水平以及抗炎/促炎因子微平衡的影響,并進一步探討阿司匹林對AD的潛在防治作用及可能機制。研究方法40只雄性SD大鼠按照完全隨機的方法分為四個實驗組,10只/組。(1)對照組:自由飲用蒸餾水,喂養(yǎng)3周后,使用腦立體定向儀進行大鼠右側(cè)側(cè)腦室注射5μl的無菌生理鹽水,再給予蒸餾水喂養(yǎng)3周;自由進食。(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;自由進食。(3)低劑量阿司匹林干預組(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;自由進食。(4)高劑量阿司匹林干預組(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;自由進食。側(cè)腦室注射術(shù)后繼續(xù)喂養(yǎng)3周后,通過Morris水迷宮檢測大鼠空間學習、記憶能力;采用雙抗體夾心ELISA法檢測海馬區(qū)組織中IL-1β、TNF-α、IL-4和IL-10的表達水平;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)空間學習記憶能力:與對照組比較,AD模型大鼠空間學習記憶能力顯著下降(P0.001),1mg/ml Asp組大鼠與對照組差異有統(tǒng)計學意義(P0.05),2mg/ml Asp組大鼠與對照組比較無統(tǒng)計學意義(P0.05);與AD模型組比較,1mg/ml Asp組大鼠與模型組差異有統(tǒng)計學意義(P0.05),2mg/ml Asp組大鼠與模型組差異有統(tǒng)計學意義(P0.01);Asp干預組組間比較,差異無統(tǒng)計學意義(P0.05)。(2)海馬區(qū)IL-1β表達:與對照組比較,AD模型組大鼠海馬組織中IL-1β表達水平升高顯著(P0.001),Asp干預組與對照組差異均無統(tǒng)計學意義(P0.05);與AD模型組比較,低、高劑量Asp干預組IL-1β表達水平均明顯下降,差異均有統(tǒng)計學意義(P0.01);Asp干預組組間比較差異無統(tǒng)計學意義(P0.05)。(3)海馬區(qū)TNF-α表達:與對照組比較,模型組大鼠海馬組織中TNF-α表達水平顯著升高(P0.001),1mg/ml Asp組與對照組差異有統(tǒng)計學意義(P0.01),2mg/ml Asp組與對照組差異無統(tǒng)計學意義(P0.05);Asp干預組與AD模型組比較,2mg/ml Asp組大鼠海馬組織中TNF-α表達水平明顯降低(P0.01),而1mg/ml Asp組與AD模型組差異無統(tǒng)計學意義(P0.05);Asp干預組組間比較差異無統(tǒng)計學意義(P0.05)。(4)海馬區(qū)IL-4表達:與對照組大鼠比較,AD模型組大鼠海馬組織中IL-4表達水平下降顯著(P0.001),1mg/ml Asp組與對照組差異有統(tǒng)計學意義(P0.01),2mg/ml Asp組與對照組差異無統(tǒng)計學意義(P0.05);與AD模型組比較,2mg/ml Asp組大鼠海馬區(qū)組織中IL-4表達水平明顯升高,差異有統(tǒng)計學意義(P0.01),1mg/ml Asp組與AD模型組差異無統(tǒng)計學意義(P0.05);Asp干預組組間比較,差異無統(tǒng)計學意義(P0.05)。(5)海馬區(qū)IL-10表達:與對照組比較,AD模型組大鼠海馬區(qū)組織中IL-10表達水平下降顯著(P0.01),1mg/ml Asp組與對照組差異無統(tǒng)計學意義(P0.05),2mg/ml Asp組與對照組差異無統(tǒng)計學意義(P0.05);與模型組比較,2mg/ml Asp組大鼠海馬區(qū)組織中IL-10表達水平明顯升高(P0.05),1mg/ml Asp組大鼠與AD模型組差異無統(tǒng)計學意義(P0.05);Asp干預組組間比較,差異無統(tǒng)計學意義(P0.05)。(6)海馬區(qū)NF-κB和i NOS陽性表達:與對照組比較,AD模型組海馬區(qū)組織中NF-κB和i NOS陽性表達明顯顯著增加,神經(jīng)元缺失明顯,而Asp干預組海馬區(qū)組織中均有不同程度的NF-κB、i NOS陽性表達和神經(jīng)元缺失,其中2mg/ml Asp干預組與對照組大致相當。(7)海馬區(qū)神經(jīng)元:與對照組比較,AD模型組海馬區(qū)神經(jīng)元缺失明顯,而Asp干預組海馬區(qū)均有不同程度神經(jīng)元缺失,其中2mg/ml Asp干預組神經(jīng)元缺失不明顯,與對照組大致相當。研究結(jié)論阿司匹林干預可保護AD模型大鼠空間學習記憶能力;阿司匹林通過抑制炎性反應、NF-κB通路活化及i NOS表達,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,進而調(diào)整促炎/抗炎因子失衡狀態(tài),在AD發(fā)病過程中抑制神經(jīng)炎性反應發(fā)展,發(fā)揮抗炎、保護神經(jīng)元和學習記憶能力的作用。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.16

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