本文選題：瘦素 + 阿爾茨海默病　； 參考：《中國人民解放軍醫(yī)學(xué)院》2013年碩士論文

【摘要】：阿爾茨海默病(Alzheimer’s disease, AD)是一種神經(jīng)退行性疾病,以神經(jīng)纖維纏結(jié)(neurofibrillary tangle, NFT)和老年斑(senile plaque, SP)為主要病理特征,其中神經(jīng)纖維纏結(jié)的本質(zhì)是過度磷酸化的tau堆積。AD的病因和發(fā)病機(jī)制迄今尚不清楚,臨床上也缺乏有效藥物,因此有關(guān)發(fā)病機(jī)制以及新的藥物作用靶點(diǎn)的探索,一直是研究的熱點(diǎn)。 瘦素(Leptin)是一種中樞性能量代謝調(diào)節(jié)因子，在炎癥反應(yīng)和創(chuàng)傷修復(fù)方面也具有重要功能,有關(guān)其神經(jīng)保護(hù)作用的研究也引起廣泛關(guān)注。為了解leptin在擬AD樣損傷中的作用及其分子機(jī)制,本研究擬用岡田酸（okadaicacid, OA）作為誘導(dǎo)劑建立神經(jīng)母細(xì)胞瘤細(xì)胞系（SH-SY5Y）AD模型、原代海馬神經(jīng)元AD模型及器官型腦片AD模型,探討leptin對擬AD樣損傷的調(diào)節(jié)及其作用機(jī)制,并在AD患者尸檢腦組織切片中觀察病理改變。 本研究包括下述4個部分：第一部分Leptin通過抑制Cdk5(細(xì)胞周期蛋白依賴性蛋白激酶5)在神經(jīng)母細(xì)胞瘤細(xì)胞系tau過度磷酸化中發(fā)揮的保護(hù)作用；第二部分Leptin在原代海馬神經(jīng)元tau過度磷酸化中的神經(jīng)保護(hù)作用及可能的分子機(jī)制；第三部分Leptin在器官型腦片擬AD樣模型中發(fā)揮的神經(jīng)保護(hù)作用；第四部分AD患者尸檢腦組織海馬區(qū)病理觀察及檢測。結(jié)果如下： 1在SH-SY5Y細(xì)胞系水平上建立擬AD樣細(xì)胞模型 （1） OA作為誘導(dǎo)劑處理細(xì)胞,可成功建立tau蛋白過度磷酸化的擬AD樣細(xì)胞模型；400ng/mL leptin處理細(xì)胞6h顯著增加細(xì)胞活力，說明leptin對細(xì)胞有保護(hù)作用； （2）免疫熒光雙染法顯示，ObR與Cdk5、ObR與p-tau均存在共表達(dá),提示ObR與二者之間均有可能存在相互作用； （3） leptin能夠劑量依賴性地降低p-tau蛋白水平，并顯著下調(diào)Cdk5（導(dǎo)致tau蛋白過度磷酸化的直接因素）表達(dá), ObR特異性封閉劑則可以逆轉(zhuǎn)上述作用，說明leptin與其受體結(jié)合通過抑制Cdk5表達(dá)來降低p-tau水平； （4）SiRNA沉默Cdk5表達(dá)可降低p-tau,而Cdk5的化學(xué)抑制劑Roscovitine則不能達(dá)到這一效果。 2在原代海馬神經(jīng)元水平上建立擬AD樣細(xì)胞模型 （1）AD主要病變區(qū)域集中在海馬組織，該部分使用原代海馬神經(jīng)元旨在表現(xiàn)leptin對病變位置細(xì)胞的保護(hù)作用。模型建立后, ObRb mRNA表達(dá)顯著升高,提示AD模型中l(wèi)eptin可能通過其長型受體發(fā)揮效應(yīng)； （2）與細(xì)胞系水平一致,在原代神經(jīng)元中l(wèi)eptin顯著降低Cdk5及p-tau表達(dá), ObR特異性封閉劑則可以逆轉(zhuǎn)上述作用；SiRNA使Cdk5沉默可降低p-tau,而Cdk5化學(xué)抑制劑Roscovitine則不能； （3） OA誘導(dǎo)模型后ERK1/2（細(xì)胞外調(diào)節(jié)蛋白激酶1/2）過度激活,而leptin可以下調(diào)其活化；利用ERK的特異性抑制劑U0126,則可以下調(diào)Cdk5及p-tau；說明ERK1/2在leptin對擬AD樣損傷的保護(hù)作用中發(fā)揮了重要作用； （4） Cdk5的抑制劑Roscovitine上調(diào)ERK1/2的活化,在3h-6h期間達(dá)到頂峰,6h以后至12h開始降低，這可以解釋其為何不能降低p-tau水平。 3在器官型腦片水平上建立擬AD樣組織模型 （1）器官型腦片屬組織水平，能夠更好的模擬組織中細(xì)胞間的相互影響，更貼近體內(nèi)環(huán)境。Real-time PCR顯示, OA誘導(dǎo)后, ObRb mRNA表達(dá)顯著升高； （2）與細(xì)胞水平一致, leptin顯著降低p-tau及Cdk5表達(dá), ObR特異性封閉劑則可以逆轉(zhuǎn)上述作用；Cdk5SiRNA處理可降低p-tau,而Cdk5抑制劑Roscovitine則不能。 4AD患者尸檢腦組織病理改變 （1）尸檢組織病理切片能夠很直觀地觀察到AD患者海馬組織的病變情況，，結(jié)果最為真實。HE染色顯示,細(xì)胞排列紊亂,可見大量細(xì)胞變性、壞死,胞漿疏松,有明顯空泡樣顆粒變性,空泡內(nèi)細(xì)胞體積明顯縮小； （2）免疫熒光雙染法顯示,病變區(qū)域ObR與Cdk5、ObR與p-tau存在共定位且熒光強(qiáng)度均較高,而正常區(qū)域則均較低； （3）免疫組化結(jié)果顯示, ObR在病變區(qū)域中分布較廣泛,而正常組織則較少。Cdk5、p-tau蛋白陽性細(xì)胞多分布于空泡狀結(jié)構(gòu),正常組織中分布較少。 綜上所述,本研究表明：（1）外源性leptin能夠通過與其長型受體ObRb結(jié)合,在擬AD樣tau過度 磷酸化損傷中發(fā)揮神經(jīng)保護(hù)作用；（2） Leptin在OA誘導(dǎo)的AD模型中,顯著降低Cdk5表達(dá),降低tau過度 磷酸化水平；（3） AD發(fā)生過程中, ERK1/2過度激活,外源性leptin可以下調(diào)其活化；（4）抑制ERK1/2可以起到下調(diào)Cdk5及p-tau水平的作用；（5） SiRNA直接干擾Cdk5基因可以起到下調(diào)p-tau的作用,而其化學(xué)抑 制劑由于會激活ERK1/2不能達(dá)到這一效果。（6） AD患者尸檢腦組織病理切片中可見,由于內(nèi)源性leptin含量較低, ObR、Cdk5、p-tau在病變區(qū)域表達(dá)增加,后兩者導(dǎo)致顯著病理改變。
[Abstract]:Alzheimer 's disease (AD) is a neurodegenerative disease with neurofibrous tangles (neurofibrillary tangle, NFT) and senile plaques (senile plaque, SP) as the main pathological features, in which the essence of neurofibrillary tangles is over phosphorylation of tau accumulation.AD. The etiology and pathogenesis are not yet clear. There is also lack of effective drugs. Therefore, exploration of pathogenesis and targets of new drugs has always been a hot topic.
Leptin (Leptin) is a central energy metabolic regulator, which also plays an important role in the inflammatory response and wound repair. The study of its neuroprotective effect has also attracted wide attention. In order to understand the role and molecular mechanism of leptin in the quasi AD like injury, this study is to use okadaicacid (OA) as an inducer. The AD model of the neuroblastoma cell line (SH-SY5Y), the AD model of the primary hippocampal neurons and the AD model of the organotypic brain slices were used to investigate the regulation and mechanism of leptin on the quasi AD like damage, and to observe the pathological changes in the brain tissue section of the autopsy of AD patients.
This study includes the following 4 parts: Part 1: the protective effect of Leptin by inhibiting the overphosphorylation of Cdk5 (cyclin dependent protein kinase 5) in the neuroblastoma cell line tau, and the neuroprotective effect and possible molecular mechanism of second part of Leptin in the excessive phosphorylation of tau in the primary hippocampal neurons; The three part of the neuroprotective effect of Leptin in the quasi AD like model of organotypic brain slices; the pathological observation and detection of the hippocampus in the fourth part of the AD patients. The results are as follows:
1 establish a quasi AD like cell model on the level of SH-SY5Y cell line.
(1) as the inducer to treat the cells, OA could successfully establish the tau protein overphosphorylated pseudo AD like cell model, and 400ng/mL leptin treatment cell 6h significantly increased the cell viability, indicating that leptin has a protective effect on the cells.
(2) immunofluorescence double staining showed that ObR and Cdk5, ObR and p-tau all co expressed, suggesting that there might be interactions between ObR and the two.
(3) leptin can reduce the level of p-tau protein in a dose-dependent manner, and significantly downregulate the expression of Cdk5 (the direct factor leading to overphosphorylation of tau protein). ObR specific sealant can reverse the above effect, indicating that leptin and its receptor are combined to reduce the level of p-tau by inhibiting the expression of Cdk5.
(4) SiRNA silencing Cdk5 expression can reduce p-tau, while Cdk5 chemical inhibitor Roscovitine can not achieve this effect.
2 Establishment of a quasi AD like cell model at the level of primary hippocampal neurons.
(1) the main lesion area of AD is concentrated in the hippocampus. This part uses the primary hippocampal neurons to show the protective effect of leptin on the lesion location cells. After the model is established, the expression of ObRb mRNA is significantly elevated, suggesting that leptin may play an effect through its long type receptor in the AD model.
(2) in accordance with the cell line level, leptin significantly reduced Cdk5 and p-tau expression in the primary neurons, and ObR specific sealants could reverse the above effect; SiRNA made Cdk5 silent to reduce p-tau, while Cdk5 chemical inhibitor Roscovitine was not.
(3) ERK1/2 (extracellular regulated protein kinase 1/2) is overactivated after OA induced model, while leptin can down regulate its activation. Cdk5 and p-tau can be downregulated by ERK specific inhibitor U0126, indicating that ERK1/2 plays an important role in the protection of leptin for quasi AD samples.
(4) the inhibitor Roscovitine of Cdk5 up-regulated the activation of ERK1/2, reached the peak during 3h-6h, and began to decrease from 6h to 12h, which could explain why it did not reduce the p-tau level.
3 Establishment of a quasi AD like tissue model at the level of organ type brain slices.
(1) organotypic brain slices belong to the level of tissue, which can better simulate the intercellular interaction in the tissue, and be closer to the.Real-time PCR in the body. After OA induction, the expression of ObRb mRNA is significantly increased.
(2) in accordance with cell level, leptin significantly reduced the expression of p-tau and Cdk5, and ObR specific sealants could reverse the above effect; Cdk5SiRNA treatment could reduce p-tau, while Cdk5 inhibitor Roscovitine did not.
Pathological changes of brain tissue at autopsy of 4AD patients
(1) the pathological sections of the autopsy can be observed to the pathological changes of the hippocampal tissue in AD patients. The results showed that the most true.HE staining showed that the cells were in disorder, and a large number of cells were denatured, necrotic, cytoplasm was loose, there were obvious vacuolar degeneration, and the volume of cells in the vacuoles decreased obviously.
(2) immunofluorescence double staining showed that CO location and fluorescence intensity of ObR and Cdk5, ObR and p-tau were higher in lesion areas than in normal areas.
(3) the results of immunohistochemical staining showed that ObR was widely distributed in the lesion area, while the normal tissue was less.Cdk5, and the p-tau protein positive cells were mostly distributed in the vacuolated structure, and the normal tissues were less distributed.
To sum up, this study shows that: (1) exogenous leptin can combine with its long receptor ObRb to produce excessive AD like tau.
Neuroprotective effect was observed in phosphorylation injury; (2) Leptin significantly reduced Cdk5 expression and reduced tau overexpression in OA induced AD model.
Phosphorylation level; (3) during the occurrence of AD, ERK1/2 excessively activates and exogenous leptin can down regulate its activation; (4) inhibition of ERK1/2 can play the role of down regulation of Cdk5 and p-tau; (5) SiRNA directly interferes with Cdk5 gene which can play the role of downregulation of p-tau, and its inhibition is inhibited.
(6) the pathological sections of brain tissue of AD patients showed that the endogenous leptin level was low because of the low level of endogenous leptin.
The expression of ObR, Cdk5 and p-tau increased in the lesion area, and the latter two resulted in significant pathological changes.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R749.16

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