水通道蛋白4介导星形胶质细胞对脑内源性及外源性β-淀粉样蛋白的反应

发布时间：2018-04-21 18:02

本文选题：阿尔兹海默病 + β-淀粉样蛋白　；参考：《南京医科大学》2013年硕士论文

【摘要】：研究背景:近年来研究表明星形胶质细胞在阿尔兹海默病(AD)的病理进程中发挥重要作用。水通道蛋白4(AQP4)作为重要的胶质细胞水转运蛋白具有调控星形胶质细胞生物学特性和功能的作用。我们前期研究发现AQP4基因敲除影响了β-淀粉样蛋白(Aβ)对培养的星形胶质细胞的毒性反应,但AQP4在AD进程中星形胶质细胞反应性病理变化中的作用有待于进一步研究。研究目的:本学位论文拟研究、阐明AQP4在星形胶质细胞对内源性及外源性Aβ病理反应性过程中的调节作用,为进一步探索以AQP4为靶标的AD治疗策略提供基础理论和实验依据。研究方案和内容:(1)为了明确AQP4在星形胶质细胞对外源性Aβ反应性增生中的作用,成年AQP4基因敲除和野生型小鼠大脑皮质微量注射Aβ1-42,48 h后灌注取材,行HE染色和TUNEL、胶质细胞纤维酸性蛋白(GFAP)、AQP4、低密度脂蛋白受体相关蛋白-1(LRP-1)免疫组织化学染色,观察星形胶质细胞活化和神经元凋亡。(2)为了明确AQP4是否参与星形胶质细胞对脑内源性Aβ聚集的反应性变化,APP/PS1转基因及同窝野生型小鼠饲养至3、6、11月龄,硫磺素S荧光染色观察不同月龄小鼠Aβ斑块积累的情况;GFAP、AQP4或LRP-1免疫组织化学染色结合刚果红组织化学染色观察星形胶质细胞活化情况、AQP4及Aβ转运体的表达变化。(3)为了进一步明确AQP4在APP/PS1转基因小鼠脑内星形胶质细胞反应性增生中的作用,应用AQP4基因敲除小鼠与APP/PS1转基因小鼠杂交构建、获得AQP4基因敲除APP/PS1转基因小鼠,饲养至3、6月龄,行硫磺素S染色和GFAP、AQP4、LRP-1、离子钙接头蛋白1(Iba-1)或突触素(SYN)免疫组织化学结合刚果红染色,观察其斑块的积累情况以及星形胶质细胞与小胶质细胞的反应性变化。结果:(1)同野生型小鼠相比,AQP4基因敲除鼠对外源性Aβ1-42诱导的星形胶质细胞反应性活化程度明显降低,而脑组织损伤和细胞凋亡加重;注射部位皮层AQP4及LRP-1表达上调,提示AQP4基因缺失降低了星形胶质细胞对外源性Aβ的活化反应和清除,从而加剧了Aβ对神经元的损害。(2)3月龄APP/PS1转基因小鼠脑内偶见Aβ斑块,但至6月龄起,出现Aβ斑块明显沉积;斑块周围星形胶质细胞活化伴随AQP4和LRP-1的表达明显升高,提示AQP4参与了星形胶质细胞对Aβ斑块的病理反应过程以及Aβ清除转运。(3)同APP/PS1小鼠相比,AQP4基因缺失APP/PS1小鼠于3月龄时脑内星形胶质细胞活化不明显,6月龄时,反应性星形胶质细胞增生明显增加,而脑内Aβ斑块沉积减少,但斑块周围的LRP-1表达、小胶质细胞活化以及SYN丢失无明显差异,提示AQP4基因缺失特异性影响了AD模型鼠脑内Aβ聚集过程中星形胶质细胞病理反应性变化。结论:上述实验结果证实AQP4介导了星形胶细胞对内、外源性Aβ的反应性变化,AQP4缺失后延缓了星形胶细胞对Aβ反应性病理进程。
[Abstract]:Background: recent studies have shown that astrocytes play an important role in the pathogenesis of Alzheimer's disease (AD). Aquaporin 4 (AQP4) plays an important role in regulating the biological characteristics and functions of astrocytes as an important glial cell water transporter. Our previous studies have found that AQP4 knockout affects the toxicity of 尾 -amyloid protein A 尾 to cultured astrocytes, but the role of AQP4 in the pathological changes of astrocyte reactivity in AD process needs further study. Objective: to elucidate the regulatory role of AQP4 in the process of endogenous and exogenous A 尾 pathological reactivity of astrocytes in order to provide theoretical and experimental basis for the further exploration of AD therapy strategy targeting AQP4. In order to clarify the role of AQP4 in astrocyte response to exogenous A 尾, adult AQP4 knockout and intracerebral cortex microinjection of A 尾 1-42U 48 h later were perfused. He staining, Tunel staining, glial fibrillary acidic protein GFAPP4, low density lipoprotein receptor-associated protein (LRP-1) immunohistochemical staining were used. In order to determine whether AQP4 is involved in the reactivity of astrocytes to endogenous A 尾 aggregation in the brain, APP / PS1 transgenic mice and wild-type mice in the same litter were fed to the age of 11 months. Observation of the accumulation of A 尾 plaques in mice with sulfur S fluorescence staining and immunohistochemical staining of GFAPAP AQP4 or LRP-1 combined with Congo Red histochemical staining to observe the expression of Astrocyte Activation and A 尾 Transporter In order to further clarify the role of AQP4 in the reactive proliferation of astrocytes in the brain of APP/PS1 transgenic mice, AQP4 gene knockout mice and APP/PS1 transgenic mice were used to construct AQP4 gene knockout APP/PS1 transgenic mice. The plaque accumulation and the reactivity of astrocytes to microglia were observed by immunohistochemical staining with sulfur S staining, GFAP AQP4 LRP-1, ion calcium junction protein 1 (Iba-1) or synaptophysin (SYN) with Congo red staining. Results compared with wild-type mice, the reactive activation of astrocytes induced by exogenous A 尾 1-42 was significantly decreased, the brain injury and apoptosis were aggravated, and the expressions of AQP4 and LRP-1 were up-regulated in cortex. It was suggested that the deletion of AQP4 gene decreased the activation and clearance of astrocytes to exogenous A 尾, and aggravated the damage of A 尾 to neurons. Occasionally, A 尾 plaques were seen in the brain of APP/PS1 transgenic mice at the age of 2 months, but the A 尾 plaques were obviously deposited in the brain of APP/PS1 transgenic mice at the age of 6 months. The activation of astrocytes around plaque was accompanied by a marked increase in the expression of AQP4 and LRP-1. The results suggest that AQP4 is involved in the pathological reaction of astrocytes to A 尾 plaques, and A 尾 clearance and transport. 3) compared with APP/PS1 mice, the activation of astrocytes in the brain of APP/PS1 mice with deletion of APP/PS1 gene is not obvious at the age of 6 months. Reactive astrocyte proliferation increased significantly, but A 尾 -plaque deposition decreased, but there was no significant difference in LRP-1 expression, microglial activation and SYN loss around the plaque. These results suggest that the deletion of AQP4 gene specifically affects the pathological reactivity of astrocytes during A 尾 aggregation in the brain of AD model. Conclusion: these results confirm that AQP4 mediates the reactivity of astrocytes to internal and external A 尾 and delays the pathological process of astrocytes reactivity to A 尾 after the deletion of AQP4.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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