【摘要】：阿爾茨海默病(Alzheimer disease, AD)多在老年時期起病，起病隱秘，病程緩慢時間長且不可逆，是一組目前病因并不明確的腦部原發(fā)性退行性變性疾病。臨床上以學習記憶認知能力進行性衰退為主，常伴有一定程度的語言、精神和人格等方面的異常。而其基本病理特點表現(xiàn)為Aβ沉積形成的老年斑、磷酸化Tau蛋白沉積形成的神經(jīng)纖維纏結以及大量膽堿能神經(jīng)元的丟失等。由于AD病人發(fā)病影響因素較多，病因病理機制非常復雜，故目前治療并無明顯突破，缺乏有效安全的治療藥物與手段。 課題組人員前期已經(jīng)發(fā)現(xiàn)人參皂苷Rd能夠抑制AD模型鼠海馬內(nèi)炎癥反應的發(fā)生，抑制培養(yǎng)的海馬神經(jīng)元氧化應激損傷而保護神經(jīng)元。故本課題以此為研究基礎，從體內(nèi)、體外、純體外三方面觀察Rd對實驗性AD模型的神經(jīng)保護作用并對其機制作初步探討，為開發(fā)安全有效的防治AD新藥提供基礎研究依據(jù)。 研究目的：探討人參皂苷Rd對實驗性AD模型的學習記憶、病理特征的影響及對其保護機制的初步探討，為尋找防治AD藥物以及研究Rd作用靶點提供一定的實驗基礎。 研究方法：（1）研究對象：急性腦損傷模型：成年清潔級SD大鼠雙側海馬CA1區(qū)立體定向微量注射Aβ1-40；慢性AD動物模型：APP轉(zhuǎn)基因小鼠；體外細胞模型：Aβ25-35干預的皮層神經(jīng)元。（2）對急性腦損傷模型大鼠和APP轉(zhuǎn)基因小鼠進行Morris水迷宮檢測觀察Rd對AD模型動物的學習記憶行為能力的影響，Nissl染色觀察其組織形態(tài)學變化；對Aβ25-35干預的皮層神經(jīng)元進行MTT染色觀察Rd對其存活率的影響和進行免疫熒光染色觀察細胞形態(tài)學變化。（3）Western blot方法檢測磷酸化Tau蛋白主要磷酸化位點的表達，觀察Rd對AD模型內(nèi)病理特征的影響，，同時對調(diào)控磷酸化Tau蛋白的關鍵激酶和磷酸酶的表達也進行了觀察。初步探討Rd對AD模型動物的保護作用機制。（4）體外純化學實驗進一步排除各種體內(nèi)體外環(huán)境的影響，Western blot方法檢測化學反應后磷酸化Tau蛋白主要磷酸化位點表達的變化，進一步探討Rd對AD模型動物的保護的作用機制及其作用靶點。 實驗結果：（1）Rd（5，10，30mg/kg）對Aβ1-40急性腦損傷大鼠的學習記憶功能均有改善:能夠縮短海馬微量注射Aβ1-40大鼠水迷宮逃避潛伏期的時間、增加模型鼠穿越原平臺次數(shù)，減少模型鼠海馬CA1區(qū)神經(jīng)元的丟失；Rd（10mg/kg）可明顯改善APP轉(zhuǎn)基因小鼠的學習記憶功能：縮短APP轉(zhuǎn)基因小鼠水迷宮逃避潛伏期的時間、增加模型鼠穿越原平臺次數(shù)；Rd（2.5，5μM）能提高體外培養(yǎng)皮層神經(jīng)元的活性；（2）Rd能夠減少各模型組磷酸化Tau蛋白的表達，抑制GSK-3β的表達，提高PP-2A的活性；（3）體外純化學實驗結果表明Rd能夠減少化學反應后各磷酸化位點Tau蛋白的表達。 實驗結論：人參皂苷Rd增強了實驗性AD模型動物的學習記憶能力，提高了AD細胞模型的存活率。其機制可能為Rd能抑制GSK-3β的表達與活性，提高PP-2A的活性，從而減少過度磷酸化Tau蛋白的形成與沉積，發(fā)揮其對AD模型的神經(jīng)保護作用。
[Abstract]:Alzheimer's disease (AD) is a kind of primary degenerative disease of the brain which is not clear in the old age. It is mainly characterized by the progressive decline of the cognitive ability of learning and memory, often accompanied by some degree of abnormality in the aspects of language, spirit and personality. The basic pathological features of the present invention are age spots formed by A-type deposition, the entanglement of the nerve fibers formed by the phosphorylation of the Tau protein, and the loss of a large number of cholinergic neurons. Due to the high incidence of AD patients, the pathogenesis and the pathological mechanism are very complex, so the current treatment has no obvious breakthrough, and the effective and safe treatment medicine and the method are lacking. The early stage of the research group has found that the ginseng soap and the Rd can inhibit the occurrence of inflammatory reaction in the hippocampus of the AD model, inhibit the oxidative stress damage of the cultured hippocampal neurons, and protect the nerves. In this paper, the neuroprotective effect of Rd on experimental AD (AD) model and its mechanism were studied in vivo, in vitro and in vitro, and a basic study was provided for the development of a safe and effective anti-AD new drug. The purpose of this study was to study the effects of ginseng soap and the effect on the learning and memory and the pathological characteristics of the experimental AD model and the preliminary discussion of its protective mechanism, and to provide some practical measures for finding the target of preventing and treating AD and studying the target of Rd. Foundation of study: (1) Study object: (1) Study object: model of acute brain injury: three-dimensional directional microinjection of A-1-40, chronic AD animal model: APP transgenic mouse; in vitro cell model: A-25-35 intervention Cortical neurons. (2) The effect of Rd on learning and memory capacity of AD model animals was observed by Morris water maze test in rats and APP transgenic mice of acute brain injury model. The effect of Rd on the survival rate and the immunofluorescent staining of the cortical neurons in A-25-35 were observed by MTT staining. (3) The expression of the main phosphorylation sites of the phosphorylated Tau protein was detected by Western blot, and the effect of Rd on the pathological characteristics of the AD model was observed. A preliminary study was made to protect the animal from AD model. (4) The effect of Rd on the expression of the main phosphorylation sites of the phosphorylated Tau protein after chemical reaction was further determined by the in vitro pure chemical experiment, and the mechanism of the protective effect of Rd on the animal in AD model was further discussed. The results showed that (1) Rd (5,10,30 mg/ kg) improved the learning and memory function of A-1-40 rats with acute brain injury: the time to avoid the incubation period of the water maze of the rats with A-1-40 in the hippocampus was shortened, the number of the model mice crossing the original platform was increased, and the hippocampal CA1 of the model rats was reduced. The loss of zone neurons; Rd (10 mg/ kg) can improve the learning and memory function of the APP transgenic mice: shorten the time of the water maze of the APP transgenic mice to avoid the incubation period, and increase the number of the model mice crossing the original platform; and Rd (2.5,5. mu.M) can improve the in vitro culture skin. and (2) Rd can reduce the expression of phosphorylated Tau protein in each model group, inhibit the expression of GSK-3 antigen, and improve the activity of PP-2A; (3) in vitro pure chemical experiment results show that Rd can reduce the phosphorylation sites T after chemical reaction The results of the experiment: The ginseng soap and the Rd enhance the learning and memory ability of the experimental AD model animals and improve the A. The survival rate of the D-cell model can be increased by the mechanism of which the expression and the activity of the GSK-3 antigen can be inhibited by Rd, and the activity of the PP-2A is improved, so that the formation and the deposition of the over-phosphorylated Tau protein can be reduced, and the response to the AD
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R285.5;R749.16

【引證文獻】

相關博士學位論文 前1條

1 孔令提;人參皂苷元的藥代動力學研究[D];北京協(xié)和醫(yī)學院;2013年

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