本文選題：阿爾茨海默病　切入點(diǎn)：ApoE4　出處：《福建醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：【目的】過(guò)度磷酸化的tau蛋白是阿爾茨海默病(Alzheimer’s disease,AD)的病理特征神經(jīng)元纖維纏結(jié)的主要組成成分。而目前普遍認(rèn)為,ApoE中的ε4等位基因已經(jīng)成為了AD最為主要的危險(xiǎn)相關(guān)基因,且參與了AD的各種病理過(guò)程。到目前為止,學(xué)者們對(duì)apoE4與Aβ的關(guān)系已經(jīng)進(jìn)行了廣泛的研究,然而apoE4與tau蛋白磷酸化的關(guān)系仍不是很清楚。故本文擬從在體和體外兩個(gè)方面進(jìn)行探討。在體,用人源的APOE基因(ε3和ε4)置換小鼠的APOE基因,然后與apoE-/-/5xFAD轉(zhuǎn)基因小鼠進(jìn)行交配,從而培育出ApoE/5xFAD(簡(jiǎn)稱EFAD)轉(zhuǎn)基因小鼠。通過(guò)對(duì)EFAD轉(zhuǎn)基因小鼠進(jìn)行研究,擬在AD的遺傳背景下觀察內(nèi)源性apoE的不同基因型(apoE3和apoE4)對(duì)5xFAD轉(zhuǎn)基因小鼠的認(rèn)知功能、tau蛋白磷酸化水平以及神經(jīng)纖維形態(tài)的影響,并探討引起tau蛋白磷酸化改變的可能上游機(jī)制。體外,通過(guò)對(duì)表達(dá)Aβ的N2a/APP695細(xì)胞株給予外源性apoE肽段及相關(guān)抑制劑干預(yù),進(jìn)一步證實(shí)在體所觀察到的tau蛋白磷酸化及機(jī)制。最終為AD的治療及新藥的開發(fā)提供一定的理論依據(jù)。【方法】1、在體部分,本實(shí)驗(yàn)采用SPF級(jí)3月齡及7月齡雄性E3FAD轉(zhuǎn)基因小鼠和E4FAD轉(zhuǎn)基因小鼠,每組9-12只。首先,通過(guò)Morris水迷宮檢測(cè)不同月齡不同APOE基因型轉(zhuǎn)基因小鼠學(xué)習(xí)記憶能力是否存在差異。然后,通過(guò)改良的Marsland氏銀染方法對(duì)大腦皮層神經(jīng)纖維進(jìn)行形態(tài)學(xué)染色,觀察不同月齡APOE的不同基因型對(duì)小鼠神經(jīng)纖維形態(tài)的影響;其次,通過(guò)Western blot和免疫組化的方法探索EFAD轉(zhuǎn)基因小鼠大腦皮層的taupT205、taupS396、taupS404和taupT231的磷酸化水平及總tau的水平。接下來(lái),通過(guò)Western blot的方法對(duì)tau蛋白磷酸化的相關(guān)激酶GSK3β和CDK5及其調(diào)節(jié)亞單位P35、P25的表達(dá)水平進(jìn)行檢測(cè)。最后,通過(guò)Western blot的方法對(duì)calpain-CDK5信號(hào)通路中calpain的表達(dá)情況進(jìn)行了檢測(cè)。2、體外部分,對(duì)穩(wěn)定表達(dá)突變型APP695的小鼠神經(jīng)母細(xì)胞瘤細(xì)胞株(N2a/APP695)予以200nM的外源性重組人apoE3和apoE4肽段干預(yù)24小時(shí),因此實(shí)驗(yàn)分為apoe3干預(yù)組、apoe4干預(yù)組和溶媒對(duì)照組。首先,采用westernblot的方法對(duì)不同組taupt205、taups396、taups404和taupt231的磷酸化水平進(jìn)行檢測(cè),驗(yàn)證動(dòng)物實(shí)驗(yàn)中所觀察到的現(xiàn)象是否能在體外進(jìn)行模擬。然后,對(duì)tau蛋白激酶cdk5及其調(diào)節(jié)亞單位p35、p25的表達(dá)水平進(jìn)行檢測(cè),同時(shí)進(jìn)一步應(yīng)用cdk5抑制劑進(jìn)行干預(yù),進(jìn)一步在體外確認(rèn)是否cdk5影響tau蛋白磷酸化。最后,對(duì)calpain的表達(dá)水平進(jìn)行檢測(cè),從而證實(shí)其中所包含的機(jī)制。【結(jié)果】1、在體實(shí)驗(yàn)結(jié)果顯示:a.行為學(xué)結(jié)果表明:在3月齡時(shí),e3fad轉(zhuǎn)基因小鼠與e4fad轉(zhuǎn)基因小鼠之間的學(xué)習(xí)記憶能力無(wú)明顯統(tǒng)計(jì)學(xué)差異;而在7月齡時(shí),與e3fad轉(zhuǎn)基因小鼠相比,e4fad轉(zhuǎn)基因小鼠學(xué)習(xí)記憶能力明顯下降,表現(xiàn)在前5天訓(xùn)練時(shí)尋找平臺(tái)的潛伏期明顯延長(zhǎng);第6天平臺(tái)探索實(shí)驗(yàn)中,在平臺(tái)所在象限停留時(shí)間明顯縮短、穿越原平臺(tái)所在位置的次數(shù)明顯減少。b.marsland氏銀染結(jié)果顯示:在3月齡時(shí),e3fad轉(zhuǎn)基因小鼠與e4fad轉(zhuǎn)基因小鼠的皮層神經(jīng)纖維均比較完整,且走行很規(guī)律,但e4fad轉(zhuǎn)基因小鼠的神經(jīng)纖維稍增粗、深染,提示e4fad轉(zhuǎn)基因小鼠的神經(jīng)纖維已有輕微損害。然而,在7月齡時(shí),e3fad轉(zhuǎn)基因小鼠的神經(jīng)纖維仍然完整且走行規(guī)律,但e4fad轉(zhuǎn)基因小鼠的神經(jīng)纖維已明顯破壞,表現(xiàn)為神經(jīng)纖維的斷裂且排列的雜亂無(wú)序。c.westernblot和組化結(jié)果顯示:與e3fad轉(zhuǎn)基因小鼠相比,早在3月齡時(shí),e4fad轉(zhuǎn)基因小鼠大腦皮層taupt205的磷酸化水平明顯增加;到7月齡時(shí),e4fad轉(zhuǎn)基因小鼠taupt205、taups396、taups404和taupt231的磷酸化水平均明顯增加。且e4fad轉(zhuǎn)基因小鼠腦內(nèi)cdk5、p35、p25及calpian明顯增高,但gsk3β活性在兩組之間無(wú)明顯的統(tǒng)計(jì)學(xué)差異。2、體外實(shí)驗(yàn)結(jié)果表明:a.給予n2a/app695細(xì)胞株200nm外源性重組人apoe3和apoe4肽段干預(yù)后,apoe4組taupt205、taups396、taups404和taupt231的磷酸化水平明顯增加;cdk5、p35、p25及calpian的表達(dá)量也明顯增加。b.在給予cdk5抑制劑后,taupt205、taups396、taups404和taupt231的磷酸化水平明顯下降�！窘Y(jié)論】1、apoe4所導(dǎo)致的tau蛋白磷酸化水平的增加早于學(xué)習(xí)記憶能力的下降。2、在5xfad轉(zhuǎn)基因小鼠中,tau蛋白磷酸化水平的增加是由于cdk5活性升高所引起,而與GSK3β無(wú)關(guān)。3、ApoE4能夠通過(guò)calpain-CDK5信號(hào)通路介導(dǎo)tau蛋白的過(guò)度磷酸化,從而破壞神經(jīng)元的骨架結(jié)構(gòu),最終導(dǎo)致5xFAD轉(zhuǎn)基因小鼠的認(rèn)知功能損害。
[Abstract]:[Objective] hyperphosphorylation of tau protein of Alzheimer's disease (Alzheimer 's disease, AD) a major component of the pathological characteristics of neurofibrillary tangles. But it is generally believed that the ApoE 4 allele AD has become the most dangerous gene related major, and participate in a variety of pathological processes AD so far, the scholars on the relationship between the apoE4 and A beta have been widely studied, but the relationship between apoE4 and tau phosphorylation is not very clear. Therefore, this paper discusses from two aspects in vivo and in vitro. In vivo, APOE gene of human origin (E 3 and E 4 APOE) gene replacement in mice, and apoE-/-/5xFAD transgenic mice were mated, to develop a ApoE/5xFAD (EFAD) transgenic mice. Through the study of EFAD transgenic mice to different genotypes observed endogenous apoE in the genetic background of AD (under APO E3 and apoE4) on the cognitive function of 5xFAD transgenic mice, tau protein phosphorylation and morphology of nerve fibers, and to explore the causes may change the phosphorylation of tau upstream mechanism. In vitro by N2a/APP695 cells on the expression of A beta exogenous apoE peptide inhibitors and related intervention, further confirmed that tau protein phosphorylation and mechanism of the observed in vivo. Finally developed for treatment and new drug AD to provide a theoretical basis. [Methods] 1, in part, this experiment adopts 3 month old grade SPF and 7 month old male E3FAD transgenic mice and E4FAD transgenic mice, 9-12 rats in each group. First, the ability of learning and memory by Morris water maze test in different months in different genotypes of APOE transgenic mice and whether there are differences. Then, the morphology of nerve fibers in the cerebral cortex stained by Marsland's modified silver staining to observe the different Effect of different genotypes of APOE month old mice on the fiber morphology of nerve; secondly, by Western blot and immunohistochemistry of EFAD transgenic mouse brain cortex taupT205, taupS396, taupS404 and taupT231 phosphorylation level and total tau level. Then, based on tau phosphorylation by Western blot correlation GSK3 and CDK5 kinase beta and its regulatory subunit P35, detect the expression level of P25. Finally, through the method of Western blot expression of calpain in calpain-CDK5 signaling pathway were detected by.2, in part, to the stable expression of murine neuroblastoma cell lines with mutant APP695 (N2a/APP695) 200nM to be exogenous recombinant human apoE3 and apoE4 peptide for 24 hours, so the experiment was divided into apoE3 treatment group, apoE4 intervention group and control group. Firstly, using the Westernblot method to the different groups of taupt20 5, taups396, taups404 were detected and the phosphorylation level of taupt231, whether the observed animal verification experimental phenomena can be simulated in vitro. Then, the tau protein kinase Cdk5 and its regulatory subunit p35, detect the expression level of p25, and further application of Cdk5 inhibitor intervention, further confirm whether Cdk5 in vitro effect of Tau protein phosphorylation. Finally, detect the expression level of calpain, thus confirming mechanism contained. [results] 1, the in vivo results showed that A. behavioral results showed that: in the 3 month old, the ability of learning and memory between e3fad transgenic mice and e4fad transgenic mice had no significant difference; while in 7 month old, compared with e3fad transgenic mice, e4fad transgenic mice significantly decreased learning and memory ability, performance in the first 5 days of training significantly prolonged the latency to find the platform balance; Sixth Taiwan exploration experiment, shorten the residence time in the platform quadrant and frequency of crossing the original platform location significantly reduced.B.marsland's silver staining results showed that: in 3 month old, cortical nerve fibers in e3fad transgenic mice and e4fad transgenic mice were relatively complete, and walking regularly, but the e4fad transgenic mice nerve fibers increased slightly coarse, dark stained, suggesting that e4fad transgenic mice nerve fibers have been slight damage. However, in 7 month old, e3fad transgenic mice nerve fibers still intact and running rules, but the e4fad transgenic mice nerve fiber has obvious damage, is broken and arranged out of order.C.westernblot and the immunohistochemical results showed: compared to the nerve fiber with e3fad transgenic mice, as early as 3 month old, e4fad transgenic mouse brain cortex taupt205 phosphorylation significantly increased to 7 month old, e4fad transgenic; Mouse taupt205, taups396, taups404 and taupt231 phosphorylation levels were significantly increased. And in the brain of e4fad transgenic mice Cdk5, p35, P25 and calpian were increased, but the GSK3 activity in the two groups had no statistically significant difference between the.2 and the experimental results show that: a. n2a/ APP695 cells 200nm exogenous recombinant human apoE3 and apoE4 peptide intervention group apoE4, taupt205, taups396, taups404 and taupt231 phosphorylation level was significantly increased; Cdk5, p35, P25 and calpian expression was significantly increased in the.B. inhibitor of Cdk5, taupt205, taups396, taups404 and taupt231 phosphorylation levels were significantly decreased. [Conclusion] 1, increase of tau the phosphorylation level of apoE4 in the early in the learning and memory ability decreased.2 in 5xfad transgenic mice, tau protein phosphorylation level increase is due to the increase of Cdk5 activity caused by.3, and has nothing to do with the GSK3 beta, ApoE4 It can mediate the excessive phosphorylation of tau protein through calpain-CDK5 signaling pathway, thereby disrupting the skeleton structure of neurons, and ultimately leading to cognitive impairment in 5xFAD transgenic mice.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R749.16

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