本文關(guān)鍵詞： 阿爾茨海默病 S14G-Humanin β-淀粉樣蛋白 Morris水迷宮 炎癥因子 免疫組織化學 ELISA 轉(zhuǎn)基因小鼠 方差分析　出處：《第四軍醫(yī)大學》2012年碩士論文　論文類型：學位論文

【摘要】：研究背景和目的： 阿爾茨海默病（Alzheimer’s disease，AD）是近年來老年人最常見的神經(jīng)系統(tǒng)變性疾病，其主要的病理學特點表現(xiàn)為形成老年斑、海馬錐體細胞的顆�？张葑冃�、神經(jīng)原纖維的纏結(jié)、軸索、突觸異常的斷裂以及神經(jīng)元的減少。該病起病比較隱匿，常伴有進行性的記憶減退和人性格的改變。到目前為止，其確切的病因和明確的發(fā)病機制尚不清楚，目前大量的研究結(jié)果顯示腦內(nèi)β-淀粉樣蛋白（amyloid-beta protein，Aβ）在腦內(nèi)聚集并且纖維化和沉積形成老年斑以及Aβ對他周圍的突觸和神經(jīng)元的毒性作用導致AD產(chǎn)生和發(fā)展。S14G-Humanin（HNG）是Humanin（HN）的合成衍生物，體外研究表明該藥品具有極強的神經(jīng)保護功能，在動物體內(nèi)初步的研究也證實HNG可有效改善側(cè)腦室注射Aβ后所導致的認知功能障礙。然而迄今為止，HNG對于成年AD轉(zhuǎn)基因小鼠已有大量Aβ斑塊沉積時的治療作用及其相關(guān)機制仍不明確，，因此，深入闡明HNG在AD發(fā)病過程中的治療作用及其相關(guān)機制有助于探索新的AD治療方法，并為其應(yīng)用提供相應(yīng)的實驗基礎(chǔ)和理論依據(jù)。本項實驗我們將通過應(yīng)用九月齡的阿爾茨海默病APPswe/PS1dE9轉(zhuǎn)基因動物模型（APPswe/PS1dE9轉(zhuǎn)基因小鼠），使用HNG療法治療3月，通過評定其行為學以及腦內(nèi)的不同種類Aβ和膠質(zhì)細胞及炎癥細胞的變化進行相關(guān)分析，進一步闡明HNG療法對已有大量Aβ斑塊沉積的AD癥的療效及相關(guān)機制。 研究方法： 本項實驗以九月齡成年APPswe/PS1dE9雌鼠和與之相匹配的常規(guī)同窩野生鼠（WT）為對象，隨機分為以下4組（每組8只小鼠）：APPswe/PS1dE9+HNG治療組，APPswe/PS1dE9+生理鹽水治療組，野生型+HNG治療組，和野生型+生理鹽水治療組。分別給予HNG和等體積的生理鹽水腹腔注射治療3個月，然后通過Morris水迷宮，免疫組織化學，熒光染色和ELIST等方法檢測小鼠的空間學習，記憶能力及不同種類Aβ和膠質(zhì)細胞及炎癥細胞的變化，并應(yīng)用相關(guān)統(tǒng)計學方法深入探討HNG在AD中的治療作用及其相關(guān)機制。 研究結(jié)果： （1）Morris水迷宮試驗定位航行實驗中，與WT+生理鹽水治療組和WT+HNG治療組相比，APPswe/PS1dE9+生理鹽水治療組的潛伏期明顯延長（P0.001），而與APPswe/PS1dE9+生理鹽水治療組相比，APPswe/PS1dE9+HNG治療組的潛伏期顯著縮短（P0.01）。在Morris水迷宮空間探索實驗中，與WT+生理鹽水治療組和WT+HNG治療組相比，APPswe/PS1dE9+生理鹽水治療組在目標象限停留時間明顯縮短（P0.001），而與APPswe/PS1dE9+生理鹽水治療組相比，APPswe/PS1dE9+HNG治療組在目標象限停留時間顯著延長（P0.001）。 （2）使用免疫組織化學和Th-S熒光染色方法分別檢測APPswe/PS1dE9+生理鹽水治療組和APPswe/PS1dE9+HNG治療組小鼠腦內(nèi)Aβ斑塊和纖維性Aβ斑塊沉積情況。與APPswe/PS1dE9+生理鹽水治療組相比，APPswe/PS1dE9+HNG治療組小鼠腦內(nèi)皮層中Aβ斑塊沉積面積及纖維性Aβ斑塊沉積面積的百分比均顯著減少（P0.001），海馬中Aβ斑塊沉積面積及纖維性Aβ斑塊沉積面積的百分比也顯著減少（P0.01）。 （3）使用ELISA方法檢測APPswe/PS1dE9+生理鹽水治療組和APPswe/PS1dE9+HNG治療組小鼠腦內(nèi)可溶性和不可溶性Aβ含量的變化情況。與APPswe/PS1dE9+生理鹽水治療組相比，APPswe/PS1dE9+HNG治療組小鼠腦內(nèi)皮層和海馬中總的不可溶性Aβ含量（P0.001）、不可溶性Aβ1-40含量（P0.01）和不可溶性Aβ1-42含量（P0.001）均顯著下降，而腦內(nèi)皮層和海馬中總的可溶性Aβ含量、可溶性Aβ_(1-40)含量和可溶性Aβ1-42含量均無顯著差異（P0.05）。 （4）使用ELISA方法檢測APPswe/PS1dE9+生理鹽水治療組和APPswe/PS1dE9+HNG治療組小鼠腦內(nèi)APP βCTF含量的變化情況。與APPswe/PS1dE9+生理鹽水治療組相比，APPswe/PS1dE9+HNG治療組小鼠腦內(nèi)皮層和海馬中APP βCTF含量均無顯著差異（P0.05）。 (5)使用免疫組織化學方法檢測各處理組小鼠腦內(nèi)皮層和海馬中反應(yīng)性星形膠質(zhì)細胞和活化小膠質(zhì)細胞的增生情況。與WT+生理鹽水治療組和WT+HNG治療組相比，APPswe/PS1dE9+生理鹽水治療組小鼠皮層和海馬中檢測到的反應(yīng)性星形膠質(zhì)細胞的數(shù)量以及活化小膠質(zhì)的細胞數(shù)量都是明顯增加的（P0.001），而與APPswe/PS1dE9+生理鹽水治療組相比，APPswe/PS1dE9+HNG治療組小鼠皮層和海馬中的反應(yīng)性星形膠質(zhì)細胞數(shù)量和活化小膠質(zhì)細胞數(shù)量均明顯減少（P0.001） （6）使用ELISA方法測量各處理組小鼠腦中炎性細胞因子IL-1β、IL-6和TNFα的含量變化并分析各組之間的差異。與WT+生理鹽水治療組和WT+HNG治療組相比，APPswe/PS1dE9+生理鹽水治療組小鼠腦內(nèi)IL-1β含量（P0.001）、IL-6含量（P0.01）和TNFα含量（P0.001）均顯著增加，與APPswe/PS1dE9+生理鹽水治療組相比，APPswe/PS1dE9+HNG治療組小鼠腦內(nèi)IL-1β含量（P0.001）， IL-6含量（P0.01）和TNFα含量（P0.001）含量均顯著顯著減少。 研究結(jié)論： 1．腹腔注射HNG可以有效改善成年APPswe/PS1dE9轉(zhuǎn)基因小鼠的認知功能障礙。 2．HNG可以顯著抑制成年APPswe/PS1dE9轉(zhuǎn)基因小鼠腦內(nèi)Aβ斑塊和纖維性Aβ斑塊的沉積。 3．HNG可以顯著減少成年APPswe/PS1dE9轉(zhuǎn)基因小鼠腦內(nèi)總的不可溶性Aβ含量、不可溶性Aβ_(1-40)含量和不可溶性Aβ1-42含量。 4．HNG可以顯著抑制成年APPswe/PS1dE9轉(zhuǎn)基因小鼠腦內(nèi)小膠質(zhì)細胞和星形膠質(zhì)細胞的活化和增生。 5．HNG可以顯著減少成年APPswe/PS1dE9轉(zhuǎn)基因小鼠腦內(nèi)IL-1β、IL-6和TNFα等炎性反應(yīng)因子的含量
[Abstract]:Background and purpose of the study : Alzheimer ' s disease ( AD ) is one of the most common neurodegenerative diseases in the elderly in recent years . The main pathological features of this disease are the formation of senile plaques , the vacuolation of pyramidal cells in hippocampus , the occurrence and development of neurons . Study method : In this experiment , the following four groups ( 8 mice in each group ) were randomly divided into 4 groups ( 8 mice in each group ) : the following 4 groups ( 8 mice in each group ) : the treatment group , the treatment group , the wild type + HNG treatment group , the wild type + HNG treatment group , the wild type + HNG treatment group , and the wild type + physiological saline treatment group . The spatial learning , memory capacity and the changes of different kinds of A尾and glial cells and inflammatory cells were detected by Morris water maze , immunohistochemistry , fluorescence staining and ELIST , and the treatment effect of HNG in AD and related mechanisms were discussed in detail . Results of the study : ( 1 ) In the Morris water maze test , compared with the WT + NS treatment group and WT + HNG treatment group , the latency of the treatment group was significantly shortened compared with the treatment group of WT + NS and WT + HNG ( P0.01 ) . ( 2 ) Using immunohistochemical and Th - S fluorescence staining methods , the percentage of A尾 plaque deposition area and fibrous A 尾 plaque deposition in the brain cortex of the mice were significantly decreased ( P0.01 ) compared with that in the control group , and the percentage of A尾 plaque deposition area and the fibrous A beta plaque deposition area in the hippocampus were significantly decreased ( P0.01 ) . ( 3 ) The content of soluble A尾 _ ( 1 - 40 ) and soluble A - 尾 _ ( 1 - 40 ) in the brain cortex and hippocampus of the mice were significantly lower than those in the control group ( P < 0.01 ) , but there was no significant difference between the contents of soluble A尾1 - 40 and soluble A尾1 - 42 in the brain cortex and hippocampus ( P0.05 ) . ( 4 ) There was no significant difference in the content of APP - 尾 CTF in the brain of the mice treated with the treatment group and the control group in the treatment group of APPS1d9 + NS + HNG treated by ELISA . The contents of APP - 尾 CTF in the brain cortex and hippocampus of the mice treated with APPS1d9 + HNG were not significantly different ( P < 0.05 ) . ( 5 ) The proliferation of reactive astrocytes and activated microglial cells in the cerebral cortex and hippocampus of each treatment group was detected by immunohistochemistry . The number of reactive astrocytes and the number of activated microglial cells in the cortex and hippocampus of the mice were significantly increased compared with those in the WT + NS treatment group and the WT + HNG treatment group ( P0.001 ) . ( 6 ) The contents of IL - 1尾 , IL - 6 and TNF偽 in brain of mice were measured by ELISA . The contents of IL - 1尾 , IL - 6 ( P0.01 ) and TNF - 偽 ( P0.001 ) in brain were significantly increased compared with those in WT + NS treatment group and WT + HNG treatment group . Conclusions of the study : 1 . Abdominal injection of HNG can effectively improve the cognitive impairment of the transgenic mice of the adult APGwe / PS1d9 . 2 . HNG can significantly inhibit the deposition of A尾plaques and fibrous A beta plaques in the brains of adult APPSl / PS1d9 transgenic mice . 3 . HNG could significantly reduce the content of insoluble A尾in the brain of the transgenic mice , and the content of insoluble A 尾 _ ( 1 - 40 ) and the content of insoluble A尾1 - 42 in the brains of the transgenic mice . 4 . HNG can significantly inhibit the activation and proliferation of microglial cells and astrocytes in the brains of the transgenic mice of the adult APGwe / PS1d9 . 5 . HNG can significantly reduce the levels of inflammatory response factors such as IL - 1尾 , IL - 6 , and TNF偽 in the brain of the transgenic mice of the adult APGwe / PS1d9 .

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R749.16

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