瘦素经Cdk5途径改善阿尔茨海默病样tau蛋白过度磷酸化的研究

发布时间：2018-05-26 15:47

本文选题：瘦素 + 阿尔茨海默病　；参考：《中国人民解放军医学院》2013年硕士论文

【摘要】：阿尔茨海默病(Alzheimer’s disease, AD)是一种神经退行性疾病,以神经纤维缠结(neurofibrillary tangle, NFT)和老年斑(senile plaque, SP)为主要病理特征,其中神经纤维缠结的本质是过度磷酸化的tau堆积。AD的病因和发病机制迄今尚不清楚,临床上也缺乏有效药物,因此有关发病机制以及新的药物作用靶点的探索,一直是研究的热点。瘦素(Leptin)是一种中枢性能量代谢调节因子，在炎症反应和创伤修复方面也具有重要功能,有关其神经保护作用的研究也引起广泛关注。为了解leptin在拟AD样损伤中的作用及其分子机制,本研究拟用冈田酸（okadaicacid, OA）作为诱导剂建立神经母细胞瘤细胞系（SH-SY5Y）AD模型、原代海马神经元AD模型及器官型脑片AD模型,探讨leptin对拟AD样损伤的调节及其作用机制,并在AD患者尸检脑组织切片中观察病理改变。本研究包括下述4个部分：第一部分Leptin通过抑制Cdk5(细胞周期蛋白依赖性蛋白激酶5)在神经母细胞瘤细胞系tau过度磷酸化中发挥的保护作用；第二部分Leptin在原代海马神经元tau过度磷酸化中的神经保护作用及可能的分子机制；第三部分Leptin在器官型脑片拟AD样模型中发挥的神经保护作用；第四部分AD患者尸检脑组织海马区病理观察及检测。结果如下： 1在SH-SY5Y细胞系水平上建立拟AD样细胞模型（1） OA作为诱导剂处理细胞,可成功建立tau蛋白过度磷酸化的拟AD样细胞模型；400ng/mL leptin处理细胞6h显著增加细胞活力，说明leptin对细胞有保护作用；（2）免疫荧光双染法显示，ObR与Cdk5、ObR与p-tau均存在共表达,提示ObR与二者之间均有可能存在相互作用；（3） leptin能够剂量依赖性地降低p-tau蛋白水平，并显著下调Cdk5（导致tau蛋白过度磷酸化的直接因素）表达, ObR特异性封闭剂则可以逆转上述作用，说明leptin与其受体结合通过抑制Cdk5表达来降低p-tau水平；（4）SiRNA沉默Cdk5表达可降低p-tau,而Cdk5的化学抑制剂Roscovitine则不能达到这一效果。 2在原代海马神经元水平上建立拟AD样细胞模型（1）AD主要病变区域集中在海马组织，该部分使用原代海马神经元旨在表现leptin对病变位置细胞的保护作用。模型建立后, ObRb mRNA表达显著升高,提示AD模型中leptin可能通过其长型受体发挥效应；（2）与细胞系水平一致,在原代神经元中leptin显著降低Cdk5及p-tau表达, ObR特异性封闭剂则可以逆转上述作用；SiRNA使Cdk5沉默可降低p-tau,而Cdk5化学抑制剂Roscovitine则不能；（3） OA诱导模型后ERK1/2（细胞外调节蛋白激酶1/2）过度激活,而leptin可以下调其活化；利用ERK的特异性抑制剂U0126,则可以下调Cdk5及p-tau；说明ERK1/2在leptin对拟AD样损伤的保护作用中发挥了重要作用；（4） Cdk5的抑制剂Roscovitine上调ERK1/2的活化,在3h-6h期间达到顶峰,6h以后至12h开始降低，这可以解释其为何不能降低p-tau水平。 3在器官型脑片水平上建立拟AD样组织模型（1）器官型脑片属组织水平，能够更好的模拟组织中细胞间的相互影响，更贴近体内环境。Real-time PCR显示, OA诱导后, ObRb mRNA表达显著升高；（2）与细胞水平一致, leptin显著降低p-tau及Cdk5表达, ObR特异性封闭剂则可以逆转上述作用；Cdk5SiRNA处理可降低p-tau,而Cdk5抑制剂Roscovitine则不能。 4AD患者尸检脑组织病理改变（1）尸检组织病理切片能够很直观地观察到AD患者海马组织的病变情况，，结果最为真实。HE染色显示,细胞排列紊乱,可见大量细胞变性、坏死,胞浆疏松,有明显空泡样颗粒变性,空泡内细胞体积明显缩小；（2）免疫荧光双染法显示,病变区域ObR与Cdk5、ObR与p-tau存在共定位且荧光强度均较高,而正常区域则均较低；（3）免疫组化结果显示, ObR在病变区域中分布较广泛,而正常组织则较少。Cdk5、p-tau蛋白阳性细胞多分布于空泡状结构,正常组织中分布较少。综上所述,本研究表明：（1）外源性leptin能够通过与其长型受体ObRb结合,在拟AD样tau过度磷酸化损伤中发挥神经保护作用；（2） Leptin在OA诱导的AD模型中,显著降低Cdk5表达,降低tau过度磷酸化水平；（3） AD发生过程中, ERK1/2过度激活,外源性leptin可以下调其活化；（4）抑制ERK1/2可以起到下调Cdk5及p-tau水平的作用；（5） SiRNA直接干扰Cdk5基因可以起到下调p-tau的作用,而其化学抑制剂由于会激活ERK1/2不能达到这一效果。（6） AD患者尸检脑组织病理切片中可见,由于内源性leptin含量较低, ObR、Cdk5、p-tau在病变区域表达增加,后两者导致显著病理改变。
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

【参考文献】