星形胶质细胞介导的β-淀粉样蛋白降解机制的研究

发布时间：2018-03-18 08:33

  本文选题：阿尔茨海默病　切入点：星形胶质细胞　出处：《宁波大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：通过星形胶质细胞培养和激光共聚焦成像的方法，研究阿尔茨海默病(Alzheimer’s disease, AD)的模型鼠——APPswe/PS1ΔE9转基因鼠以及老年星形胶质细胞溶酶体对可溶性β-淀粉样蛋白(β-Amyloid protein42，Aβ42)的降解能力是否下降。探讨细胞内PH改变是否影响星形胶质细胞溶酶体对外源性Aβ42的降解能力。通过星型胶质细胞溶酶体对Aβ42降解机制的研究，进一步了解AD可能的发病机制，为寻找AD的治疗靶点提供理论和实验依据。 方法：通过PCR的方法鉴定APPswe/PS1ΔE9双转基因鼠，，取新生第一天的小鼠进行原代星形胶质细胞的培养。用带绿色荧光的Aβ42(Hilyte Fluor488-labled Amyloid-β protein42，Aβ42)处理体外培养第7天(day7in vitro, DIV7)，体外培养第21天(day21in vitro,DIV21)的星形胶质细胞，孵育30min。用l μM的溶酶体的染料Lysotracker DND Red99(DND-99)对溶酶体染色，激光共聚焦显微镜观察，分析APP/PS1双转基因鼠星形胶质细胞溶酶体与Aβ42共定位率和正常组比是否有差别。同时分析DIV7星形胶质细胞内Aβ42和溶酶体的荧光强度随时间变化情况；观察不同组星形胶质细胞内吞Aβ42的动态变化过程。用NH4Cl处理改变细胞内PH后，分析AD组星形胶质细胞溶酶体与外源性Aβ42共定位率是否有差异。 结果： 1.星形胶质细胞内吞外源性Aβ42后30min，定位于溶酶体。 2.经Aβ42和DND-99孵育30min后，Aβ42和溶酶体的荧光强度在DIV7星形胶质细胞AD组和正常组间均没有差异，说明AD组和正常组DIV7星形胶质细胞对Aβ42的内吞没有差异。随着时间变化，AD组DIV7星形胶质细胞溶酶体与外源性Aβ42的共定位率与正常组比均显著偏高，37℃孵育2h后， AD组DIV7星形胶质细胞内Aβ42荧光强度高于正常组，AD组DIV7星形胶质细胞内溶酶体荧光强度低于正常组。 3. AD组DIV21星形胶质细胞溶酶体和Aβ42的荧光强度与正常组DIV21星形胶质细胞比均下降，正常组DIV21星形胶质细胞溶酶体和Aβ42的荧光强度与正常组DIV7比均下降。 4.经25mM的NH4Cl溶液处理后，AD组星形胶质细胞溶酶体与Aβ42的共定位率下降。 结论： 1. DIV7星形胶质细胞内吞外源性Aβ42后30min定位于溶酶体。 2. AD小鼠DIV7星形胶质细胞溶酶体对外源性Aβ42的降解能力下降。 3. DIV21老胶质细胞对外源性Aβ42的内吞能力下降，DIV21老星形胶质细胞溶酶体活性下降。 4. NH4Cl能加强星形胶质细胞溶酶体对外源性Aβ42的降解。
[Abstract]:Objective: to study the methods of astrocyte culture and laser confocal imaging. To study the degradation of soluble 尾 -amyloid protein (尾 -Amyloid protein 42 A 尾 42) by APPsweP PS1 螖 E9 transgenic mice and senile astrocyte lysosomes in Alzheimer's disease (ADD) model mice. To investigate whether the changes of intracellular PH affect the degradation of soluble 尾 -amyloid protein A 尾 42.To investigate the effects of intracellular PH changes on the degradation of soluble 尾 -amyloid protein (尾 -Amyloid protein 42A 尾 42) in mice with Alzheimer's disease (ADD). The degradation of exogenous A 尾 42 by lysosomes in astrocytes, and the mechanism of degradation of A 尾 42 by astrocyte lysosomes. To further understand the possible pathogenesis of AD and to provide theoretical and experimental evidence for finding therapeutic targets for AD. Methods: APPswe/PS1 螖 E9 transgenic mice were identified by PCR. Primary astrocytes were cultured in mice on the first day of birth. Astrocytes were cultured in vitro with A 尾 42 Fluor488-labled Fluor488-labled A myloid- 尾 42A 尾 42) on the 7th day of culture, and on the 21st day of culture with day21in Vitro-DIV21), and the astrocytes were cultured on the 21st day in vitro, and the astrocytes were cultured on day 7 and day 21 respectively, and the astrocytes were cultured in vitro with A 尾 42n Hilyte Fluor488-labled Amyloid- 尾 42 (A 尾 42). The astrocytes were cultured in vitro on the 7th day. After incubation for 30 min, the lysosomes were stained with 1 渭 M lysosomal dye Lysotracker DND Red99 DND-99, and observed by confocal laser microscopy. To analyze whether the co-localization rate of lysosome and A 尾 42 of APP/PS1 double transgenic mice is different from that of normal group, and to analyze the change of fluorescence intensity of A 尾 42 and lysosome in DIV7 astrocytes with time. To observe the dynamic changes of endocytosis A 尾 42 in astrocytes of different groups. After treating with NH4Cl to change the intracellular PH, we analyzed whether there were differences in the co-localization rate of lysosomes between astrocytes and exogenous A 尾 42 in AD group. Results:. 1. After endocytosis of A 尾 42, astrocytes were located in lysosomes for 30 min. 2. After incubation with A 尾 42 and DND-99 for 30 minutes, the fluorescence intensity of A 尾 42 and lysosome was not different between AD group and normal group of DIV7 astrocytes. The results showed that the uptake of A 尾 42 by DIV7 astrocytes in AD group and normal group was different, and the co-localization rate of lysosome of DIV7 astrocytes and exogenous A 尾 42 in AD group was significantly higher than that in normal group after incubation at 37 鈩

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