ERβ对小鼠海马actin细胞骨架聚合与学习记忆的调节作用研究

发布时间：2018-05-16 04:44

本文选题：海马 + 雌激素　；参考：《第三军医大学》2017年硕士论文

【摘要】：研究报道随着年龄的逐渐增长,老化所引起的雌激素(estrogen,E_2)水平降低在痴呆症如阿尔兹海默病(Alzheimer’s disease,AD)的学习记忆方面扮演着重要的角色。目前,AD的具体发病机制仍未完全明确,现有的治疗方案疗效并不理想。因此,研究探索AD的发病机理和治疗方法对于预防和治疗AD,促进医疗护理和家庭照护都具有深远的影响。E_2不仅可以影响女性的生理发育,还在改善老年的记忆力衰退方面也具有积极的作用,在调控海马突触可塑性以及学习记忆的调节过程中需要借助雌激素受体(estrogen receptor,ER)的参与来产生作用。E_2的受体分为两大类:一类是膜受体GPR30,位于细胞内的膜性成分包括细胞膜、线粒体膜、内质网等,通过第二信使途径介导E_2对某些生理功能的调节;另一类是经典的核受体,包括雌激素α受体(estrogen receptorα,ERα)和雌激素β受体(estrogen receptorβ,ERβ),它们主要在细胞核内通过与DNA结合从而调节靶基因的转录。类固醇受体辅助活化因子-1(steroid receptor coactivator-1,SRC-1)作为是促进核受体调节靶基因转录活动所必需的辅助因子之一,高表达于海马、大脑皮层等脑区,其表达受产后发育和性腺切除的影响。有研究表明在给予卵巢切除手术和核受体抑制剂处理后海马突触蛋白表达和突触及树突棘密度明显下降,雌激素替代治疗及其核受体激动剂可以增加突触蛋白的表达和树突棘的密度,但ERβ在当中的作用机制目前尚不明确。ERβ克隆于1996年,过去20年的研究发现ERβ在多种动物的体内均有较为广泛的表达,并且参与了E_2对多种脑功能作用的调节,且在不同的分区其表达分布和功能作用也不尽相同。在中枢神经系统内,大脑皮层、海马等区域有大量ERβ的表达,提示ERβ可能扮演着介导E_2调控学习记忆的功能性受体的角色。在生殖系统,睾丸、卵巢等组织器官内可检测到ERβ的存在,它可能参与E_2调节生殖细胞的生长、分化等过程。另外,在心血管、消化道等系统内也可检测到ERβm RNA,并且也具有十分重要生理意义。脑内的研究发现,利用ERβ特异性拮抗剂以及基因敲除等方法抑制ERβ后,发现海马突触蛋白表达显著下降,树突棘密度降低,出现空间学习记忆障碍。通过ERβ激动剂活化ERβ可改善突触可塑性和提高空间学习记忆,提示ERβ对学习记忆有着重要的调控作,但是其具体机制尚不清楚。肌动蛋白(actin)是构成细胞骨架的重要成分,actin聚合与解聚的动态变化是突触可塑性的基础,也是学习记忆的基础。球状肌动蛋白(即G-actin)在Profilin-1等分子的诱导下聚合成为螺旋状的肌动蛋白(F-actin)。因此,F-actin/G-actin比例的变化常作为衡量的肌动蛋白聚合程度的指标。研究还发现哺乳动物雷帕霉素靶蛋白复合体2(mammalian target of rapamycincomplex 2,m TORC2)对actin细胞骨架的聚合有着重要的调节作用,敲除m TORC2的核心成分Rictor导致actin聚合程度与树突棘密度下降,并导致严重的学习记忆障碍,而直接激活Rictor的效应分子p-AKT(AKT ser473)可逆转上述学习记忆障碍,证明m TORC2在调节actin细胞骨架聚合与学习记忆中的重要作用。既往研究报道ERβ可调节树突棘密度和学习记忆行为,但是ERβ是否调控actin细胞骨架聚合尚不清楚。目的:探讨ERβ在调控海马神经元actin细胞骨架多聚化以及学习记忆中的作用及其机制。方法:1、为了解ERβ在生后小鼠不同时相点海马的表达情况,我们运用蛋白质免疫印迹(Western blot,WB)、免疫组化(Immunohistochemistry,IHC)实验方法检测了ERβ在生后不同发育时期(P0,P7,P14,P28,P56)雌、雄小鼠海马的表达变化。2、为了探讨激活ERβ对海马actin细胞骨架聚合以及学习记忆的影响,将成年小鼠进行卵巢切除(ovariectomy,OVX),1w后在颈背部皮下注射不同剂量ERβ激动剂DPN进行预实验,分为假手术对照(Control)、卵巢切除(OVX)、OVX+1.25 mg/kg DPN、OVX+2.5 mg/kg DPN、OVX+5.0 mg/kg DPN共5组,通过检测上述处理条件下海马SRC-1、Rictor及下游靶分子p-AKT以及actin细胞骨架聚合蛋白(p-cofilin/cofilin、Profilin-1)的表达变化确定DPN处理的最佳剂量。然后,在最佳剂量(5.0 mg/kg DPN)的处理条件下,检测了DPN对OVX后上述分子表达、actin聚合状态(F-actin/G-actin比值)的变化、海马树突棘密度以及学习记忆行为变化的调控。3、取成年动情间期雌性小鼠,腹腔注射相同剂量(100ug/kg)ERβ抑制剂PHTPP,分为溶剂对照(Control)及PHTPP注射1、3、5、7d共5组,通过检测海马SRC-1、Rictor及下游靶分子p-AKT、p-cofilin/cofilin、Profilin-1的表达变化来确定PHTPP处理的最佳时间点。然后,在此最佳时间点(7d)条件下,运用Morris水迷宫方法观察小鼠空间记忆行为的变化情况,用WB、IHC以及高尔基镀银染色等方法来检测抑制ERβ活性后海马SRC-1、Rictor、p-AKT和actin细胞骨架聚合蛋白(p-cofilin/cofilin、Profilin-1)的表达变化、actin聚合状态(F-actin/G-actin比值)、海马区的树突棘密度的变化,以此来探索ERβ活性改变与学习记忆之间的相互联系。结果:1、ERβ在雌、雄小鼠P0、P7、P14、P28、P56海马中都有表达。在P0时表达高,在P7和P14时表达下降,在P28和P56时表达增加。雄性小鼠在P28时达到P0时水平,但雌性小鼠在P28时就已高于P0时的表达。2、OVX后海马SRC-1、Rictor及下游靶分子p-AKT、actin细胞骨架聚合蛋白(p-cofilin/cofilin、Profilin-1)表达均下降,1.25mg/kg DPN时仅Profilin-1的表达增加而其余分子表达无明显变化,在2.5-5.0mg/kg DPN时上述所有分子表达较OVX组上升,因此,选取5.0mg/kg为DPN的最佳剂量。Morris水迷宫发现OVX后雌性小鼠的学习记忆下降,高尔基镀银染色结果显示OVX组海马树突棘密度减少,免疫组织化学实验(immunohistochemistry,IHC)和蛋白印迹实验(Western blot,WB)显示OVX组SRC-1、Rictor及下游靶分子p-AKT、actin细胞骨架聚合调控蛋白、actin聚合状态(F-actin/G-actin比值)的表达下降,5.0mg/kg DPN处理可以逆转OVX所致的上述变化。3、海马SRC-1、Rictor、p-AKT、actin细胞骨架聚合调控蛋白(p-cofilin/cofilin、Profilin-1)在给予PHTPP注射5d时表达开始下降,除Profilin-1外其余各分子表达在7d时显著下降,因此,确定7d为PHTPP处理的最佳时间。在此条件下,Morris水迷宫发现PHTPP可致雌性小鼠的学习记忆下降,高尔基镀银染色结果显示海马树突棘密度减少,IHC和Western blot显示PHTPP下调海马SRC-1、Rictor及下游靶分子p-AKT、actin细胞骨架聚合相关蛋白、actin聚合程度(F-actin/G-actin比值)的表达。结论:1、ERβ在雌、雄小鼠生后海马内(P0~P56)均有表达,且在出生时和成年时维持在较高水平,呈现出“U-型”变化的趋势。2、激活ERβ可逆转OVX所致的海马SRC-1、Rictor及下游靶分子p-AKT、actin细胞骨架聚合蛋白表达及海马树突棘密度的下降、actin聚合状态的解聚和学习记忆行为障碍,提示活化ERβ可通过促进actin细胞骨架的聚合进而影响学习记忆行为。3、抑制ERβ可下调海马SRC-1、Rictor及下游效应分子p-AKT、actin细胞骨架聚合蛋白表达以及海马树突棘密度,促进actin聚合状态的解聚,动物出现明显的学习记忆行为障碍,提示抑制ERβ活性通过促进actin细胞骨架的解聚进而导致学习记忆行为障碍。综上所述,在本研究中,采用了卵巢切除、Morris水迷宫、IHC、Western blot以及高尔基镀银染色等多种实验技术,研究了调节ERβ活性变化对小鼠空间学习记忆行为以及SRC-1、m TORC2通路蛋白和actin细胞骨架聚合的影响,结合文献我们认为ERβ可能通过SRC-1/m TORC2调节actin细胞骨架聚合状态的变化来影响突触可塑性,最终对学习记忆行为产生影响。以上研究结果也为寻找AD预防和治疗的新靶点提供了新的实验依据。
[Abstract]:It is reported that the decrease of estrogen (E_2), which is caused by aging, plays an important role in the learning and memory of Alzheimer's disease (Alzheimer 's disease, AD). The specific pathogenesis of AD is still not complete, and the therapeutic effect of the existing treatment is not ideal. Exploring the pathogenesis and treatment of AD has a profound influence on the prevention and treatment of AD, the promotion of medical care and family care, which not only affects the physiological development of women, but also plays an active role in improving the memory decline of the aged, and in regulating the plasticity of synaptic plasticity in the hippocampus and the regulation of learning and memory. The receptors that require the participation of estrogen receptor (ER) to produce.E_2 are divided into two categories: one is the membrane receptor GPR30, and the membranous components in the cell include the cell membrane, the mitochondrial membrane, the endoplasmic reticulum, and the second messenger pathway to mediate the regulation of some physiological functions by E_2; the other is the classic nuclear receptor, including the receptor. Estrogen receptor (estrogen receptor alpha, ER alpha) and estrogen beta receptor (estrogen receptor beta, ER beta), which mainly regulate the transcription of the target gene by binding to DNA in the nucleus. Steroid receptor auxiliary activating factor -1 (steroid receptor coactivator-1, SRC-1) is essential for promoting the nuclear receptor to regulate target gene transcription activities. One of the cofactors, highly expressed in the hippocampus, cerebral cortex, and other brain regions, its expression is affected by postpartum development and gonadectomy. Studies have shown that the expression of synaptic protein and the density of synapses and dendrites of the hippocampus are significantly decreased after the treatment of ovariectomy and the treatment of nuclear receptor inhibitors. Estrogen replacement therapy and its nuclear receptor agonists can be used. The expression of synaptic protein and the density of dendritic spines are increased, but the mechanism of the role of ER beta in the.ER beta is not yet clear in 1996. The last 20 years of research have found that ER beta has been widely expressed in various animal bodies, and is involved in the regulation of E_2 on various brain functions, and the expression distribution and function in different partitions. In the central nervous system, there are a large number of ER beta expressions in the cerebral cortex and hippocampus, suggesting that ER beta may play a role in the functional receptors that mediate the learning and memory of E_2. The presence of ER beta in the reproductive system, the testis and the ovary can be detected, and it may be involved in E_2 regulation of the growth of the germ cells. In addition, ER beta m RNA can also be detected in cardiovascular, digestive tract and other systems, and it also has a very important physiological significance. In the brain, it was found that the expression of synaptic protein in the hippocampus was significantly decreased, the density of dendritic spines decreased, and spatial learning was found by using ER beta specific antagonists and gene knockout methods to inhibit ER beta. The activation of ER beta through ER beta agonists can improve synaptic plasticity and improve spatial learning and memory, suggesting that ER beta has an important regulation on learning and memory, but its specific mechanism is not yet clear. Actin (actin) is an important component of the cytoskeleton, and the dynamic changes in actin polymerization and depolymerization are the basis of synaptic plasticity. It is the basis of learning and memory. Spherical actin (G-actin) is polymerized into a spiral actin (F-actin) under the induction of Profilin-1 and other molecules. Therefore, the change in the proportion of F-actin/G-actin is often used as a measure of the degree of actin polymerization. The study also found that the mammalian rapamycin target protein complex 2 (mammalian target) Of rapamycincomplex 2, m TORC2) plays an important role in the regulation of the aggregation of the cytoskeleton of the actin. The core component of the m TORC2, Rictor, leads to a decrease in the degree of actin polymerization and dendrite density, and the cause of serious learning and memory disorders. The effect molecule p-AKT (AKT) can reverse the impairment of learning and memory. M TORC2 plays an important role in regulating the cytoskeleton polymerization and learning memory of actin. Previous studies have reported that ER beta can regulate dendrite density and learning and memory behavior, but whether ER beta regulates the aggregation of actin cytoskeleton is unclear. Objective: To explore the role of ER beta in regulating the cytoskeleton polycondensation and learning memory of hippocampal neurons in actin. Methods: 1, in order to understand the expression of ER beta in the hippocampus of different phases of the mice after birth, we used Western blot (WB) and immunohistochemistry (Immunohistochemistry, IHC) to detect the expression of ER beta in the different developmental stages (P0, P7, P14, P28, P56) and the expression of the hippocampus in male mice, in order to explore the excitation of the hippocampus. The effects of ER beta on the cytoskeleton polymerization and learning and memory of hippocampal actin cells were treated with ovariectomy (OVX) in adult mice (ovariectomy, OVX). After 1W, different doses of ER beta agonist DPN were injected subcutaneously into the back of the neck to be pre tested, divided into the sham operation control (Control), the ovariectomy (OVX), OVX+1.25 mg/kg DPN, and 5 The optimal dosage of DPN treatment was determined by detecting the expression changes of the hippocampal SRC-1, Rictor, the downstream target molecule p-AKT and the actin cytoskeleton polymeric protein (p-cofilin/cofilin, Profilin-1) under the above treatment conditions. Then, under the best dose (5 mg/kg DPN), the expression of DPN to OVX was detected and actin polymerized. The changes in the state (F-actin/G-actin ratio), the density of the hippocampal dendrites and the changes in the learning and memory behavior were regulated by.3. The adult estrus female mice were intraperitoneally injected with the same dose (100ug/kg) ER beta inhibitor PHTPP, divided into 5 groups, solvent control (Control) and PHTPP injection 1,3,5,7d, by detecting the hippocampus SRC-1, Rictor and downstream target molecule p-AKT, p-. Cofilin/cofilin, Profilin-1 expression changes to determine the best time point for PHTPP treatment. Then, under the best time point (7D), the Morris water maze method was used to observe the changes in the spatial memory behavior of mice. WB, IHC, and Golgi silver staining were used to detect the hippocampus SRC-1, Rictor, p-AKT and acti after the inhibition of ER beta activity. The changes in the expression of N cytoskeleton poly (p-cofilin/cofilin, Profilin-1), actin polymerization state (F-actin/G-actin ratio) and the density of dendritic spines in the hippocampus in order to explore the relationship between the changes of ER beta activity and learning memory. Results: 1, ER beta is expressed in female, male mice P0, P7, P14, P28, P56 hippocampus. The expression decreased at P7 and P14, and increased at P28 and P56. The male mice reached the level of P0 at P28, but the female mice were higher than P0 when P28 was higher than P0. The hippocampus SRC-1, Rictor and downstream target molecules were decreased. The expression of IN-1 was increased and the expression of other molecules was not obviously changed. The expression of all the above molecules increased at 2.5-5.0mg/kg DPN. Therefore, the optimum dose of.Morris water maze of 5.0mg/kg as DPN was selected to find the decline of learning and memory in the female mice after OVX, and the Golgi Golgi staining fruit showed that the density of the dendritic spines in the hippocampus of the hippocampus was reduced in the OVX group and the immune group was in the immune group. Immunohistochemistry (IHC) and Western blot experiments (Western blot, WB) showed that OVX group SRC-1, Rictor, and downstream target molecules p-AKT, actin cytoskeleton polymerization regulatory proteins, actin polymerization state (F-actin/G-actin ratio) decreased. T, actin cytoskeleton polymerization regulatory protein (p-cofilin/cofilin, Profilin-1) began to decrease when given PHTPP injection of 5D, and the expression of other molecules except Profilin-1 was decreased significantly. Therefore, the optimal time for 7d to be treated as PHTPP was determined. Under this condition, the Morris water maze found that the learning and memory of PHTPP to female mice decreased. The result of silver plating staining showed that the density of hippocampal dendrites decreased, and IHC and Western blot showed that PHTPP lowered the SRC-1, Rictor and downstream target molecules p-AKT, actin cytoskeleton aggregation related proteins and actin polymerization degree (F-actin/G-actin ratio). Conclusion: 1, ER beta was expressed in the hippocampus (P0~P56) in male mice after birth and at birth. At the time and in adulthood, it is maintained at a high level, showing a "U- type" change trend.2. Activation of ER beta can reverse OVX induced SRC-1, Rictor and downstream target molecules p-AKT, actin cytoskeleton polymerization protein expression and the decrease of hippocampal dendrite density, actin polymerization state depolymerization and learning memory behavior disorder, suggesting that activation ER beta can be promoted by promoting the activation of ER beta. The aggregation of the actin cytoskeleton further affects the learning and memory behavior.3, and the inhibition of ER beta can downregulate the SRC-1, Rictor and downstream effector p-AKT, the expression of the actin cytoskeleton polymerized protein and the density of the hippocampal dendrite, which promotes the depolymerization of the actin polymerization state, and the animals have obvious learning memory behavior disorders, suggesting that the inhibition of ER beta activity is promoted through the promotion of promoting the activity of ER beta. The depolymerization of the cytoskeleton into actin leads to the learning and memory behavior disorder. In this study, in this study, a variety of experimental techniques, such as ovariectomy, Morris water maze, IHC, Western blot and Golgi Golgi staining, were used in this study to study the learning and memory behavior of ER beta activity and SRC-1, m TORC2 pathway protein and act. The effect of in cytoskeleton polymerization, combined with the literature, we think that ER beta may affect the synaptic plasticity by regulating the changes in the aggregation of the actin cytoskeleton through the SRC-1/m TORC2, and ultimately affects the learning and memory behavior. The results also provide new experimental evidence for the search for new targets for the prevention and treatment of AD.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R473.74

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