内源性雌激素的神经保护作用研究

发布时间：2018-04-24 05:07

本文选题：内源性雌激素 + 雌激素受体　；参考：《第四军医大学》2014年硕士论文

【摘要】：雌激素主要由卵巢、胎盘等组织产生，参与机体各种生理和病理活动。近几十年来，，雌激素对中枢神经系统（Central Nervous System，CNS）的作用受到广泛关注。动物实验证实，雌激素不仅可以降低大脑中动脉缺血（Middle Cerebral ArteryOcclusion，MCAO）和全脑缺血（Global Cerebral Ischemia，GCI）模型的梗死容积和死亡率[1,2]，还可通过增强海马齿状回和室管膜下层的神经发生，促进小鼠中风后的恢复[3]。流行病学调查也发现，绝经前女性卒中发生率低于男性，而绝经后两者无明显差异[4,5]。高血清水平雌激素可显著提高语言工作记忆，而绝经后使用雌激素替代可使女性认知功能障碍和神经退行性疾病的发病风险减少或延缓[6]。目前认为，雌激素神经保护作用的发挥主要通过调节雌激素的合成和雌激素受体（EestrogenReceptor，ER）的表达来实现[7]，其机制包括经典的基因组信号通路、非基因组信号通路、抗氧化作用和调节线粒体功能等[8]。然而，随着研究的深入，有关雌激素神经保护作用的经典理论不断受到质疑，2003年发表在JAMA上的临床研究提示：在老年（平均年龄63岁）高危中风患者，雌激素替代疗法反而加重了中风损伤和认知功能障碍，增加了死亡率，并增加乳腺癌和子宫肌瘤的发生[9,10]；女性雌激素对中风影响试验（Women's Estrogen for StrokeTrial，WEST）结果显示，在开始的六个月致命性中风和所有早期中风发生率都有所增加[10]。雌激素的神经损伤作用被认为与其促氧化应激[11]、促炎症反应[12]和增加兴奋性毒性[13]有关。经过近年来的研究和反思，目前，研究者针对雌激素神经保护和神经损伤的双重作用提出了系列学说，包括关键期假说[14-16]、剂量依赖学说[17,18]和受体亚型特异性学说[19-21]。这些学说的建立可以部分解释雌激素产生正反两方面作用的原因。随着研究的进展，有报道指出ER的表达水平随生理周期卵巢雌激素水平的波动而变化[22]，但这种变化与脑缺血损伤之间的关系还未见报道。此外，最新报道指出哺乳动物的脑组织也能合成雌激素，其浓度和活性都远高于循环水平[23,24]，并在突触重塑中发挥着重要作用[25,26]。这些发现为雌激素的神经保护作用提供了新的思路。本研究将通过制作不同雌激素水平大鼠MCAO模型，探讨不同生理周期卵巢雌激素水平的变化对脑缺血损伤的影响及其可能的机制。并应用C57BL/6小鼠的去卵巢（Ovariectomy，OVX）和GCI模型观察海马雌激素与循环雌激素之间的关系、海马雌激素的调控方式以及其在CNS中的作用和机制。实验一不同生理周期卵巢雌激素的神经保护作用研究目的：通过构建不同雌激素水平大鼠MCAO模型，观察不同生理周期卵巢雌激素水平的变化对脑缺血损伤和预后的影响及其可能的机制。方法：将100只3月龄雌性SD大鼠分为5组（n=20）：发情前期组（Proestrus，PE组）、间期组（Diestrus，DE组）、OVX组、芳香化酶抑制剂Letrozole组（Letro组）和ER拮抗剂ICI182,780组（ICI组）。通过放射免疫法（Radioimmunoassay，RIA）检测血清17β-雌二醇（17β-Estradiol，E2）水平（n=5），采用线栓法制作大鼠MCAO模型（n=15），缺血2h，再灌注24h后进行神经功能学评分，通过TTC染色计算脑梗死容积（n=10），尼氏染色统计半暗带神经元存活情况（n=5），免疫印迹法（Western Blot，WB）分析MCAO之前相对半暗带区域皮层ERα、β蛋白表达变化（n=5）。结果： RIA检测结果显示PE时E2水平最高，DE最低；给予OVX和Letro后，大鼠E2水平明显降低；MCAO前PE组和DE组大鼠相对半暗带区域ERα蛋白表达水平无统计学差异（P0.05），但高于OVX组、Letro组和ICI组（P0.05），各组ERβ表达水平无统计学差异；MCAO后，PE组和DE组大鼠半暗带存活神经元数目无统计学差异（P0.05），但较其他三组存活神经元数目增多（P0.05），神经功能损伤程度和脑梗死容积相对其他三组也有明显改善（P0.05）。结论：正常大鼠血清E2水平随生理周期波动，PE时最高，DE时最低，给予OVX和Letro后进一步降低。不同生理周期的卵巢E2对脑缺血的损伤无明显统计学差异，都可以改善脑缺血损伤的预后，而ERα在卵巢E2介导的脑缺血损伤中可能起更关键的作用。实验二海马雌激素的神经保护作用研究目的：利用C57BL/6小鼠的OVX和GCI模型观察海马雌激素与循环雌激素之间的关系、海马雌激素的调控方式以及其在CNS中的作用和机制。方法： 100只雌性C57BL/6小鼠，随机分为5组（n=20），Control组---不做任何处理；OVX1w+Oil组---OVX1w后，芝麻油替代3w；OVX1w+E2组---OVX1w后，E2替代3w；OVX10w+Oil组---OVX10w后，芝麻油替代3w；OVX10w+E2组---OVX10w后，E2替代3w。每组动物按实验要求准备好之后，进行恐惧箱和水迷宫实验，之后每组取10只做GCI，其中5只24h后制作石蜡切片进行凋亡染色，另5只7d后制作冰冻切片进行神经元存活染色。每组剩余的10只动物，5只取血并取海马组织检测E2水平，另 5只取海马进行WB和Real-Time PCR，检测蛋白和mRNA表达水平变化。结果：本实验证实OVX后小鼠血清和海马E2水平降低，短期记忆能力和空间学习记忆能力明显受损（P0.05），GCI后凋亡神经元数目也明显增多（P0.05），而在OVX后1w进行E2替代可以提高循环和海马E2水平，明显改善依赖海马的学习记忆，减轻GCI后的神经元凋亡，但在长时间雌激素剥夺（Long-Term Estrogen Deprivation，LTED）动物E2的神经保护作用消失，产生这一现象的原因可能是海马ERα的降解和E2生成减少共同导致的。结论：本研究与“关键期假说”的观点一致。E2神经保护作用的丧失不仅与海马ERα的不可逆降解有关，还涉及海马E2生成的减少。因为海马芳香化酶表达水平在OVX10w小鼠明显减弱。芳香化酶是E2合成的关键酶，他的减少进一步导致海马E2合成的减少，此作用与ERα的降解协同，使E2的神经保护作用丧失。小结本研究证实，不同生理周期的卵巢E2和海马E2都具有明显的神经保护作用。正常大鼠血清E2水平随生理周期波动，PE时最高，DE时最低，给予OVX和Letro后进一步降低。生理周期内的卵巢E2都具有良好的神经保护作用，并能改善脑缺血损伤的预后，所以在雌性动物的脑缺血损伤实验中可以忽略生理周期变化对结果的影响，而ERα在卵巢E2介导的脑缺血损伤中可能起更关键的作用。海马E2对依赖海马的学习记忆以及神经元的存活至关重要，并可与循环E2相互作用。OVX1w后E2替代可以逆转小鼠学习记忆能力下降以及对GCI的耐受，但LTED后E2替代则无效。说明E2产生神经保护作用不仅与激素替代开始的时间有关，还与海马局部E2的产生有关，海马产生的E2在雌激素的神经保护作用中也扮演了重要的角色。
[Abstract]:In recent decades , estrogen has been widely concerned with the effects of estrogen on central nervous system ( CNS ) . In recent decades , estrogen plays an important role in central nervous system ( CNS ) . Epidemiological investigation also found that the incidence of pre - menopausal female stroke was lower than that of men , while no significant difference was found between them after menopause . High serum levels of estrogen could significantly improve the language working memory , while postmenopausal estrogen replacement could reduce or delay the risk of female cognitive dysfunction and neurodegenerative diseases . At present , estrogen receptor ( ER ) expression is mainly regulated by regulating the synthesis of estrogen and estrogen receptor ( ER ) . Its mechanism includes classical genome signal pathway , non - genomic signal pathway , anti - oxidation action and regulation of mitochondrial function .

However , with the in - depth study , the classical theory of estrogen neuroprotection has been questioned . In 2003 , the clinical study suggested that estrogen replacement therapy increased stroke damage and cognitive impairment in elderly patients with high risk stroke ( mean age 63 years ) , increased mortality , and increased the incidence of breast cancer and uterine fibroids ( 9 , 10 ) ;
Women ' s estrogen for StrokeTrial ( WEST ) showed an increase in the incidence of fatal stroke and all early stroke at the beginning of six months . The effect of estrogen on nerve injury was thought to be related to its pro - oxidative stress , the inflammatory response , the inflammatory response , and the increase of excitatory toxicity .

In recent years , the authors put forward a series of theories on the effects of estrogen on the protection of estrogen and neuroprotection .

Neuroprotective effects of estrogen on ovarian estrogen in different physiological cycles

Purpose :

The effects of different estrogen levels on cerebral ischemia injury and prognosis in rats with different estrogen levels were observed and possible mechanisms were observed .

Method :

Male SD rats were divided into 5 groups ( n = 20 ) : proestrus group ( PE group ) , interval group ( group DE ) , ovariectomized group ( DE group ) , Letrozole group ( Letro group ) and ER antagonist ICI182 , 780 group ( ICI group ) .

Results :

The results of RIA showed that E2 level was highest at PE and lowest in DE ;
The level of E2 in the rats was significantly lower than that of the rats .
There was no significant difference in the expression level of ER伪 protein between PE group and DE group ( P0.05 ) , but the expression level of ER 尾 was significantly higher than that in ovariectomized group , Letro group and ICI group ( P0.05 ) .
There was no significant difference in the number of survival neurons between PE group and DE group ( P0.05 ) , but the number of survival neurons in the other three groups increased ( P0.05 ) , and the degree of nerve function injury and the volume of cerebral infarction were significantly improved compared with other three groups ( P0.05 ) .

Conclusion :

The serum level of E2 in normal rats varies with the physiological cycle , the highest when PE is , the lowest in DE , and further decreases after the administration of ovariectomized and Letro . There is no statistical difference in the damage of E2 on cerebral ischemia in different physiological cycles .

Study on the Neuroprotective Effects of Estrogen in the Hippocampus of Experiment 2

Purpose :

The relationship between estrogen and circulating estrogen in hippocampus , the regulation of estrogen in hippocampus and its role and mechanism in CNS were observed in C57BL / 6 mice .

Method :

100 female C57BL / 6 mice were randomly divided into 5 groups ( n = 20 ) , control group - - no treatment ;
After OVX1w + Oil group - - OVX1w , sesame oil is replaced by 3w ;
After OVX1w + E2 group - - OVX1w , E2 was replaced by 3w .
After OVX10w + Oil group - - OVX10w , sesame oil is replaced by 3w ;
After OVX10w + E2 group - - OVX10w , E2 was replaced by 3w .

After each group of animals were prepared well according to the experimental requirements , a fear box and a water maze experiment were carried out , after which 10 rats were taken as the GCI , 5 hours later , paraffin sections were prepared for apoptosis staining , and another 5 days later , the frozen sections were prepared for survival staining of neurons . The remaining 10 animals in each group were taken from the blood and the hippocampus tissues were taken to detect E2 levels .

5 . The expression of protein and mRNA was detected by WB and Real - Time PCR in hippocampus .

Results :

In this experiment , the level of E2 in serum and hippocampus , short - term memory ability and spatial learning and memory ability were significantly impaired ( P0.05 ) , but the number of apoptotic neurons increased significantly after GCI ( P0.05 ) .

Conclusion :

The loss of E2 neuroprotection is not only related to the irreversible degradation of ER伪 in hippocampus , but also the decrease of the formation of E2 in hippocampus .

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The results showed that E2 and E2 in hippocampus of different physiological cycles had obvious neuroprotection function . The serum level of E2 in normal rats decreased with the physiological cycle , the lowest in DE , and decreased further after the administration of estrogen and Letro . In the experiment of cerebral ischemia injury , the effect of physiological cycle change on the outcome was ignored . The E2 substitution in the physiological cycle could reverse the memory ability of mice and the tolerance to GCI .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

【共引文献】