Aquaproin-8在卵泡发育中的作用与机制和阿尔兹海默症小鼠模型血液生物标志物研究

发布时间：2018-01-07 20:00

本文关键词：Aquaproin-8在卵泡发育中的作用与机制和阿尔兹海默症小鼠模型血液生物标志物研究　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：第一部分:Aquaporin-8在卵泡发育中的作用和机制研究水通道蛋白家族(aquaporins,AQPs)是一类在细胞膜上转运水分子的通道蛋白,在机体液体代谢中具有重要的功能。AQP8作为家族中的一员,已被证明在消化生理、肝胆生理和生殖生理中等都发挥着作用。近年来,AQP8在哺乳动物雌性生殖系统中的重要性正在被逐步揭示。本课题组曾揭示AQP8在卵巢中专一性地表达在颗粒细胞上,AQP8的缺失导致小鼠颗粒细胞水通透性降低,由此引发的生理效应是排卵数目增多和产仔数量增加,以及还出现了多卵卵泡现象。然而AQP8在卵泡发育中的作用和机制很大程度上还没被人们理解。在当前的研究里,利用AQP8基因敲除小鼠,我们分别从卵巢、卵泡、颗粒细胞三个逐渐深入的水平开展实验,发现了AQP8在卵泡发育尤其是卵泡腔形成中的新功能并阐明了相关的机理。首先我们发现AQP8敲除鼠相比于野生鼠有明显增多的有腔卵泡现象,并且通过卵泡3D体外培养的方法,我们发AQP8敲除鼠的卵泡成活率和成腔率都要明显高于野生鼠。对这一现象进行进一步分析,将野生鼠和AQP8基因敲除鼠的卵泡颗粒细胞分离、培养。在增殖实验中,FSH存在下的AQP8敲除型颗粒细胞的增殖能力相比野生型显著减弱。在横向与纵向迁移实验中,我们发现无论是否FSH的存在,AQP8敲除型卵泡颗粒细胞的迁移能力都要明显弱于野生鼠的颗粒细胞。接着我们考察了颗粒细胞的雌激素和孕激素合成能力,检测合成这两个激素的三个限速酶CYP19A1,CYP11A1和St AR的m RNA表达水平和蛋白质水平,发现两种基因型颗粒细胞激素合成能力无区别。基于这些实验结果,我们对AQP8敲除鼠有腔卵泡增多表型的机制解释是,颗粒细胞增殖和迁移能力的减弱使卵泡在腔前期更容易形成间隙和小的液体点,这些小的液体点经过聚集形成大的卵泡腔,导致AQP8敲除鼠成腔率增加。本研究为卵泡发育及卵泡腔的形成理论提供了新的视点。第二部分:阿尔兹海默症小鼠模型中血液生物标志物的研究阿尔茨海默氏病(AD)作为一种渐进性与年龄有关的神经退行性疾病,是一个全球性的健康问题。脑中Aβ42含量升高,形成Aβ纤维沉积和tau磷酸化等是导致阿尔兹海默病症的主要原因。尽管心理测试,神经影像学、基因检测和脑脊液生化检测这些手段可行,但是仍然缺乏方便、准确的预测和诊断AD的血浆生物标志物。炎性反应已经被证实是AD发病过程中一个很重要的过程,而很多文献报道炎性因子可以作为AD发病过程中的指示因子,α-2M是一种在血浆中含量丰富的血浆蛋白,在血浆中,α巨球蛋白主要作为对抗外源多肽的防御屏障。在脑中,α-2M作用是消除淀粉样斑块,通过结合Aβ,防止纤维形成和增强Aβ的清除。CFH是一种可溶性调节因子,其首要任务是补体3(C3)的调节,补体3在促炎症反应的刺激过程中起着核心作用。Clusterin(簇集蛋白)已被定位存在于淀粉样斑块中,起到抑制补体形成,结合Aβ,防止纤维形成,清除和运输Aβ跨越血脑屏障的功能。本研究旨在评价β-淀粉样蛋白,α-2巨球蛋白(α-2M),补体因子H(CFH)和成簇蛋白作为血浆生物标志物的可行性。分别用3,6,9,12月龄的APP/PS1转基因和野生型(WT)小鼠,通过Morris水迷宫实验和免疫组织化学方法对小鼠认知障碍和脑淀粉样斑块数目进行评价,结果显示APP/PS1转基因的小鼠分别在6、9和12个月呈现AD特征。然后我们用ELISA方法对不同AD进展状况的小鼠血清中Aβ40,Aβ42,α-2M,CFH和clusterin的浓度进行测定。结果显示3和6月龄转基因小鼠与对照组相比,转基因小鼠血清Aβ42水平和Aβ42/Aβ40比值显著增加。3和6月龄转基因小鼠与对照组相比,血清CFH水平显著下降。同时,12个月大的转基因小鼠与对照组相比,血清成簇蛋白水平明显升高。在转基因和野生型小鼠之间α-2M水平没有明显差异。
[Abstract]:The first part: the Aquaporin-8 study on follicular development effect and mechanism of water channel protein family (aquaporins, AQPs) is a kind of transport in the cell membrane water channel protein, the body liquid metabolism has important function of.AQP8 as a member of the family, has been shown in digestive physiology, physiology and hepatobiliary the reproductive physiology of medium played a role. In recent years, the importance of AQP8 in the female reproductive system in mammals is gradually revealed. The research group has revealed that AQP8 was specifically expressed in ovarian granulosa cells in AQP8, because of the lack of water permeability of mouse granulosa cells decreased, physiological effects which caused the increase of the number of the number of ovulation and litter increased, and also appeared in a number of egg follicles. However, phenomenon and mechanism of AQP8 on follicular development largely not understood. In the present study, the use of AQP 8 gene knockout mice, we were from the ovarian follicles, granulosa cells, three levels gradually carry out the experiment, we found AQP8 in follicular development especially the new function in the formation of follicular cavity and clarify the related mechanism. First we found that AQP8 knockout mice compared to wild-type mice have significantly increased antral follicles the phenomenon, and through the method of follicle culture 3D in vitro, we AQP8 knockout mouse follicle survival rate and cavity rate are significantly higher than the wild rats. Further analysis on this phenomenon, the wild-type mice and AQP8 knockout mouse granulosa cells cultured in isolation, proliferation. In the experiment, FSH the presence of AQP8 knockout granulosa cell proliferation was significantly reduced compared to the wild type. In the horizontal and vertical migration experiment, we found that regardless of whether the presence of FSH, AQP8 knockout migration type granulosa cells are significantly weaker than Granulosa cells in wild mice. Then we investigated the ability of granulosa cells to estrogen and progesterone synthesis, three CYP19A1 synthesis rate limiting enzyme detection of these two hormones, m RNA CYP11A1 and St AR expression and protein level, found two genotypes of granulosa cell hormone synthesis ability difference. Based on the experimental results, we the AQP8 knockout mice have the mechanism to explain antral follicle phenotype is increased, weakening of granulosa cell proliferation and migration ability of the follicles are more likely to form a small gap and the liquid in the cavity period, these small liquid after together to form large follicular cavity, resulting in AQP8 knockout mouse cavity rate increased. The research the theory provides a new perspective of follicular development and follicle cavity formation. The second part: Alzheimer's disease in a mouse model of blood biomarkers of Alzheimer's disease (AD) is a progressive and years Age related neurodegenerative disease, is a global health problem. Increased brain A beta 42 content, the formation of A beta fiber deposition and tau phosphorylation is a major cause of Alzheimer's disease. Despite the psychological test, neuroimaging, gene detection and detection of cerebrospinal fluid biochemical these means is feasible, but still the lack of convenient, plasma biomarkers predictive and accurate diagnosis of AD. Inflammatory reaction has been proved to be a very important process in the pathogenesis of AD, and many reported inflammatory factors can be used as indicator in the pathogenesis of AD, a -2M is a rich content of plasma protein in the plasma. In plasma, alpha macroglobulin as the main defense barrier against exogenous peptide. In the brain, a -2M role is to eliminate amyloid plaques, through a combination of A beta, preventing fiber formation and enhancement of A beta.CFH is a soluble regulatory clearance Its primary task is to complement factor 3 (C3) regulation, complement 3 in stimulating proinflammatory process plays a central role in.Clusterin (clusterin) has been positioned in amyloid plaques, inhibit the formation of complement, combined with the A beta, prevent fiber formation, removal and transportation across the A beta the function of the blood brain barrier. The purpose of this study was to evaluate the beta amyloid protein, alpha -2 macroglobulin (alpha -2M), complement factor H (CFH) and clusters of proteins as a plasma biomarker. The feasibility of respectively 3,6,9,12 month old APP/PS1 transgenic and wild-type (WT) mice by Morris water maze test the methods of immunohistochemistry and the number of mice with cognitive impairment and cerebral amyloid plaques were evaluated. The results showed that APP/PS1 transgenic mice showed features of AD in 6,9 and 12 months respectively. Then we use the ELISA method in serum of different AD status in A 40 beta, alpha A beta 42, -2M, C The concentration of FH and clusterin were measured. The results showed that 3 and June age transgenic mice compared with the control group, the serum of transgenic mice A beta 42 beta 40 beta 42/A level and A ratio were significantly increased in.3 and June age transgenic mice compared with the control group, the serum CFH level was significantly decreased. At the same time, the 12 month old transgenic mice compared with the control group, the serum protein levels were significantly increased. Clustering between transgenic and wild-type mice did not differ significantly between alpha -2M levels.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16;R-332

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