【摘要】：隨著人口老齡化程度的不斷加劇,一些與大腦衰老相關(guān)的神經(jīng)退行性疾病的流行程度和發(fā)病率不斷攀升,如阿爾茨海默病(Alzheimer's Disease, AD)等,日益成為威脅人類健康的主要疾病之一,也給社會(huì)、經(jīng)濟(jì)和家庭帶來沉重的負(fù)擔(dān)。AD是最常見的癡呆癥,從發(fā)現(xiàn)至今已經(jīng)一個(gè)多世紀(jì),人們對(duì)AD的成因仍不清楚,目前也沒有能有效治療AD的藥物問世。 目前FDA批準(zhǔn)的用于AD的藥物主要為基于膽堿能假說而來的膽堿酯酶抑制劑(Acetylcholinesterase inhibitor, AChE I),此類藥物通過抑制乙酰膽堿(Acetylcholine, ACh)的降解來提高大腦中的ACh水平,從而緩解AD癥狀。但隨著AD病情的加重和膽堿能神經(jīng)元的逐步丟失,病人自身產(chǎn)生的ACh會(huì)嚴(yán)重降低,因此依賴于內(nèi)源性ACh的AChE I的治療效果也會(huì)逐漸喪失。另一方面,毒蕈堿型M1乙酰膽堿受體(Muscarinic M1acetylcholine receptor, M1mAChR)在AD病人大腦中的密度沒有發(fā)生下降。而且大量研究表明,M1受體在AD的病理過程中扮演重要角色,是治療AD的一個(gè)重要靶點(diǎn)。M1受體激動(dòng)劑可以有效改善AD的多個(gè)典型癥狀,包括大腦內(nèi)由于Ap前體蛋白(Amyloid precursor protein, APP)的錯(cuò)誤剪切而造成的神經(jīng)毒性蛋白Aβ的產(chǎn)生和沉積,tau蛋白的過度磷酸化,以及由于膽堿能神經(jīng)元的丟失而造成的膽堿能系統(tǒng)的損傷等。一些M1受體激動(dòng)劑先后進(jìn)入臨床實(shí)驗(yàn),如Xanomeline在臨床實(shí)驗(yàn)中已被證明能改善AD患者的認(rèn)知能力和行為異常,但由于副作用較大而在三期臨床實(shí)驗(yàn)中被淘汰。 為了改進(jìn)Xanomeline,以期獲得活性更好、副作用更小的新型M1受體激動(dòng)劑,我們與北京大學(xué)雷小平教授課題組合作,通過對(duì)Xanomeline的母核進(jìn)行的系統(tǒng)的修飾和改造,獲得了一系列Xanomeline衍生物,本文的工作由此展開。在內(nèi)容上,本文共分為四章,從Xanomeline這個(gè)在三期臨床被淘汰的藥物出發(fā),第一章描述了對(duì)Xanomeline系列衍生物的篩選和EUK1001的發(fā)現(xiàn),并初步分析了EUK1001對(duì)APP的剪切的影響;第二章是在體實(shí)驗(yàn)部分,評(píng)價(jià)了EUK1001在AD動(dòng)物模型前腦特異性早老素雙敲除(PS cDKO)小鼠的治療效果；第三章探討了抗衰老蛋白klotho在PS cDKO小鼠神經(jīng)退行性癥狀中的可能作用；第四章則綜述了M1受體激動(dòng)劑及klotho等的一些研究進(jìn)展。具體為： 1、新型M1受體激動(dòng)(?)-Xanomeline新型衍生物EUK1001的篩選 利用基于報(bào)告基因技術(shù)的細(xì)胞篩選模型,對(duì)172個(gè)Xanomeline的衍生物進(jìn)行了篩選,找到了18個(gè)活性化合物,其中包括活性最好的兩個(gè)化合物EUK1001和EUK1002。與Xanomeline相比,EUK1001對(duì)M1受體的EC50更小,最大激活倍數(shù)更高。EUK1001和EUK1002的活性在瞬時(shí)轉(zhuǎn)染了M1受體和報(bào)告基因的中國倉鼠卵巢細(xì)胞(Chinese hamster ovary cells, CHO)細(xì)胞中得到了確證。同時(shí),克隆并在小鼠神經(jīng)母細(xì)胞瘤細(xì)胞Neuro2A (N2a)中過表達(dá)了人源APP基因瑞典型突變體(APPsw),以此為AD細(xì)胞模型,發(fā)現(xiàn)EUK1001和EUK1002能通過激活M1受體促進(jìn)具有神經(jīng)保護(hù)作用的sAPPa的釋放。從這些篩選結(jié)果和離體實(shí)驗(yàn)數(shù)據(jù)可以看出,EUK1001和EUK1002不僅可以比Xanomeline更有效地激活M1受體,還能促進(jìn)APP的α-剪切,在調(diào)節(jié)AD的Ap病理途徑中可能有一定潛力。 2、EUK1001對(duì)前腦特異性早老素雙敲除(PS cDKO) AD小鼠模型部分神經(jīng)退行性癥狀的改善 隨后的動(dòng)物藥理和毒理實(shí)驗(yàn)結(jié)果顯示,EUK1001誘發(fā)的副作用次數(shù)明顯少于Xanomeline,而EUK1002則沒有得到改進(jìn)。在本實(shí)驗(yàn)中,以早老素PS1/PS2前腦特異性雙敲除(PS cDKO)小鼠為AD動(dòng)物模型,從行為學(xué)、組織學(xué)和分子生物學(xué)等幾個(gè)不同的水平和角度,分析了EUK1001對(duì)PS cDKO小鼠的神經(jīng)退行性癥狀的影響。結(jié)果發(fā)現(xiàn),經(jīng)過3個(gè)月的慢性給藥(0.5mg/kg/day)后,在新物體識(shí)別實(shí)驗(yàn)中,EUK1001可以顯著改善PS cDKO小鼠受損的識(shí)別記憶能力,同時(shí)對(duì)大腦切片的分析結(jié)果顯示,EUK1001能有效緩解由于神經(jīng)元大量丟失而造成的腦萎縮,但在這兩方面,同劑量的Xanomeline都沒有表現(xiàn)出明顯的改善效果。最后在分子水平的分析顯示,EUK.1001和Xanomeline都能有效地降低PScDKO小鼠大腦內(nèi)的tau蛋白過度磷酸化,但它們對(duì)PS cDKO小鼠升高的炎癥反應(yīng)都沒有抑制作用。綜合這些結(jié)果可以看出,EUK1001對(duì)PS cDKO小鼠的部分神經(jīng)退行性癥狀有明顯的改善作用,而且效果要優(yōu)于同劑量的Xanomeline,具有進(jìn)一步開發(fā)的潛力。 3、前腦特異性早老素雙敲除(PS cDKO)小鼠中klotho表達(dá)的下降和血磷的升高 Klotho蛋白是一個(gè)重要的抗衰老因子,只在腎臟和大腦中高表達(dá),但以循環(huán)因子的形式在全身多個(gè)部位發(fā)揮多種功能,如調(diào)節(jié)鈣磷平衡,抗氧化應(yīng)激,抗炎癥等。Klotho水平的下降與衰老及多種疾病有關(guān),但klotho在一些神經(jīng)退行性疾病(如AD)中的作用尚未被闡明。在本實(shí)驗(yàn)中,我們分析了AD動(dòng)物模型PS cDKO小鼠中klotho的表達(dá)以及若干與klotho功能相關(guān)的指標(biāo),如血磷、血鈣和腎功能等,并且結(jié)合前人在該模型中的一些結(jié)果,初步探討了klotho在PS cDKO小鼠的神經(jīng)退行性癥狀中可能發(fā)揮的作用。結(jié)果發(fā)現(xiàn),與同齡正常對(duì)照小鼠相比,12月齡PS cDKO小鼠腎臟klotho表達(dá)顯著下降,血磷水平明顯升高,血鈣水平略微下調(diào),但沒有觀察到明顯的腎功能障礙,而在3月齡時(shí),腎臟klotho水平未發(fā)生變化,血磷血鈣亦與正常對(duì)照無異,但此時(shí)大腦klotho水平已發(fā)生下調(diào)。這些結(jié)果初步顯示,klotho可能與PS cDKO鼠的癥狀有關(guān),其表達(dá)水平的下降可能導(dǎo)致了血磷濃度的上升,但不足以引發(fā)腎功能障礙。此外,klotho作為一個(gè)抗氧化和抗炎癥的因子,其表達(dá)水平的下降可能與PS cDKO小鼠氧化壓力和炎癥反應(yīng)的升高有關(guān),但需要進(jìn)一步的實(shí)驗(yàn)證實(shí)。
[Abstract]:With the increasing aging of the population, the prevalence and incidence of some neurodegenerative diseases related to brain aging, such as Alzheimer's Disease (AD), have become one of the main diseases threatening human health, and have brought heavy burden to society, economy and family. It has been more than a century since the discovery of dementia. The cause of AD is still unknown, and there are no effective drugs to treat it.
Currently, FDA-approved drugs for AD are mainly cholinesterase inhibitors (AChE I) based on the cholinergic hypothesis. These drugs increase ACh levels in the brain by inhibiting the degradation of acetylcholine (ACh), thus alleviating AD symptoms. On the other hand, the density of muscarinic M1 acetylcholine receptor (M1mAChR) in the brain of AD patients has not decreased. And a large number of studies have shown that M1 receives. M1 receptor agonists can effectively ameliorate many typical symptoms of AD, including the production and deposition of neurotoxic protein A beta and the hyperphosphorylation of tau protein due to the wrong shearing of Ap precursor protein (APP) in the brain. Some M1 receptor agonists have been introduced into clinical trials, such as Xanomeline, which has been shown to improve cognitive and behavioral abnormalities in AD patients, but has been eliminated in phase III clinical trials due to its large side effects.
In order to improve Xanomeline and obtain a novel M1 receptor agonist with better activity and less side effects, we have obtained a series of Xanomeline derivatives through systematic modification and modification of the parent nucleus of Xanomeline in collaboration with Professor Lei Xiaoping of Peking University. In the first chapter, we describe the screening of Xanomeline derivatives and the discovery of EUK1001, and preliminarily analyze the effect of EUK1001 on the shearing of APP. In the second chapter, we evaluate the effect of EUK1001 on the forebrain specific presenilin double knockout (PS cDKO) in AD animal model in vivo. In the third chapter, the possible role of Klotho in the neurodegenerative symptoms of PS cDKO mice was discussed. In the fourth chapter, the research progress of M1 receptor agonists and Klotho was reviewed.
1, the screening of a new M1 receptor activator (-Xanomeline) new derivative EUK1001.
Using a cell screening model based on reporter gene technology, 172 derivatives of Xanomeline were screened and 18 active compounds were found, including the two most active compounds EUK1001 and EUK1002. Compared with Xanomeline, EUK1001 had smaller EC50 and higher maximum activation multiple for M1 receptor. The activities of EUK1001 and EUK1002 were transient. At the same time, we cloned and expressed a Swedish mutant of human APP gene (APPsw) in mouse neuroblastoma cell Neuro2A (N2a), which was used as AD cell model and found that EUK1001 and EUK1002 could be stimulated by stimulation. From these screening results and in vitro experimental data, we can see that EUK1001 and EUK1002 can not only activate M1 receptor more effectively than Xanomeline, but also promote alpha-shearing of APP, which may have some potential in regulating the pathological pathway of AP in AD.
2. EUK1001 improves some neurodegenerative symptoms in forebrain-specific presenilin double knockout (PS cDKO) AD mice
Subsequent animal pharmacological and toxicological experiments showed that the number of side effects induced by EUK1001 was significantly less than that induced by Xanomeline, while EUK1002 was not improved. The effects of EUK1001 on neurodegenerative symptoms in PS cDKO mice were analyzed. The results showed that EUK1001 significantly improved the impaired recognition and memory abilities in PS cDKO mice after 3 months of chronic administration (0.5mg/kg/day). The results of brain slices analysis showed that EUK1001 could effectively alleviate the impaired recognition and memory abilities in PS cDKO mice. The results showed that both EUK. 1001 and Xanomeline could effectively reduce tau protein hyperphosphorylation in the brain of PS cDKO mice, but both of them had increased inflammatory responses to PS cDKO mice. These results show that EUK1001 can obviously improve some neurodegenerative symptoms of PS cDKO mice, and the effect is better than that of Xanomeline at the same dose, which has potential for further development.
3, the decrease of Klotho expression and the increase of blood phosphorus in the forebrain specific PS cDKO mice.
Klotho protein is an important anti-aging factor, which is highly expressed only in the kidney and brain, but plays a variety of functions in the form of circulating factors in many parts of the body, such as regulating calcium and phosphorus balance, antioxidant stress, anti-inflammation and so on. In this experiment, we analyzed the expression of Klotho and some indexes related to the function of klotho, such as blood phosphorus, blood calcium and kidney function, in AD animal model PS cDKO mice, and combined with some previous results in this model, we preliminarily discussed the possible occurrence of Klotho in neurodegenerative symptoms of PS cDKO mice. The results showed that the expression of Klotho in kidney of 12-month-old PS cDKO mice was significantly lower than that of normal control mice, the level of serum phosphorus was significantly higher, and the level of serum calcium was slightly lower than that of normal control mice, but no obvious renal dysfunction was observed. These results preliminarily suggest that Klotho may be associated with the symptoms of PS cDKO mice. The decrease in Klotho expression may lead to an increase in serum phosphorus concentration but is not sufficient to induce renal dysfunction. Oxidative stress is associated with increased inflammatory response in mice, but further experimental confirmation is needed.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16;R-332

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