AD转基因小鼠小脑内突触相关蛋白及超微结构变化的实验研究

发布时间：2018-04-25 15:31

本文选题：APP/PS1转基因小鼠 + 3xTg-AD转基因小鼠　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:本研究将分别利用APPswe/PS1d E9(APP/PS1)和APPswe/PS1M146V/tau P301L(3xTg-AD)转基因小鼠模型,应用Western-blotting,免疫组织化学和透射电镜的实验手段,观察AD模型小鼠小脑内突触相关蛋白表达以及超微结构的改变,探讨AD小脑突触功能变化的可能机制,为进一步揭示小脑在AD发生中的作用提供新的理论依据。方法:选用9月龄,雄性APP/PS1双转基因小鼠、3xTg-AD基因小鼠与对照野生型C57BL/6J小鼠(wild type,WT)。将其分为野生型组(WT)、双转基因组(APP/PS1)、三转基因组(3xTg-AD)。实验动物由中国医学科学院医学实验动物研究所基因工程平台和The Jackson Laboratory公司提供。饲养环境自然昼夜节律光照,自由摄食进水,通风良好。随机将每组小鼠分成三个小组,分别进行免疫组化、Western-blotting、透射电镜实验。(1)免疫组化实验:小鼠心脏灌注,固定后取脑组织,冷冻于-80℃,随后切片进行免疫组织化学实验。脑片厚度为30μm,驴血清进行封闭,一抗4℃过夜,二抗孵育,显色,透明,封片,每步间均用PBS进行漂洗3次,每次5 min,显微镜下观察比较各组小鼠小脑皮质内Aβ斑块(6E10)、突触素(Synaptophysin)、脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)和其高亲和力受体(Tyrosine kinase B,Trk B)的表达。(2)Western-blotting实验:提取小鼠小脑组织蛋白,制备凝胶,蛋白加样后,80V电压电泳。之后转膜,BSA封闭,一抗4℃过夜,二抗孵育,最后显影曝光,用曝光仪分析电泳目的条带(Synaptophysin、BDNF、Trk-B和β-actin)的表达变化。(3)透射电镜实验:经心脏灌注、固定后取出小脑,将小脑皮质切成1mm×1mm×1mm的组织块,4%多聚甲醛固定后,用p H7.2磷酸缓冲液冲洗,置于锇酸中后固定,双蒸水冲洗。脱水,丙酮与包埋剂混合,室温浸透,环氧树脂包埋,浸透、聚合。用超薄切片机切片,醋酸铀-柠檬酸铅双染,透射电镜下观察并拍照[1],并对突触相关指标进行定量分析。结果:(1)APP/PS1组和3xTg-AD组转基因小鼠小脑皮质内均有少量Aβ斑块的沉积。(2)Western-blotting实验结果显示,与WT组相比,APP/PS1组和3xTg-AD组转基因小鼠小脑内突触素(Synaptophysin)含量明显减少(APP/PS1:P0.01,3xTg-AD:P0.01)。免疫组化实验结果显示,APP/PS1组小鼠小脑分子层(P0.01)、浦肯野细胞层(P0.01)和颗粒细胞层(P0.01)中突触素阳性表达均明显减少;3xTg-AD组分子层(P0.01)、浦肯野细胞层(P0.01)和颗粒细胞层(P0.01)中的光密度值亦明显减少,但与APP/PS1组相比,没有统计学差异(P0.05)。两组小鼠小脑内浦肯野细胞形态均无明显改变。(3)Western-blotting实验结果显示,与WT组相比,APP/PS1组和3xTg-AD组转基因小鼠小脑内,BDNF的含量均明显减少(APP/PS1:P0.01,3xTg-AD:P0.01),Trk-B含量也明显减少(APP/PS1:P0.01,3xTg-AD:P0.01)。免疫组化实验结果显示,APP/PS1组和3xTg-AD组分子层浦肯野细胞层(APP/PS1:P0.01,3xTg-AD:P0.01)和颗粒细胞层内(APP/PS1:P0.01,3xTg-AD:P0.01)阳性颗粒均明显减少,但分子层内BDNF阳性表达均无显著改变(APP/PS1:P0.05,3xTg-AD:P0.05)。APP/PS1组和3xTg-AD组小脑皮质各层内Trk-B阳性颗粒明显减少,相对光密度值在分子层(APP/PS1:P0.001,3xTg-AD:P0.001)、浦肯野细胞层(APP/PS1:P0.001,3xTg-AD:P0.001)以及颗粒细胞层(APP/PS1:P0.001,3xTg-AD:P0.001)内均明显降低。但APP/PS1组和3xTg-AD组之间没有统计学差异(APP/PS1:P0.05,3xTg-AD:P0.05)。此外,在光镜下AD小鼠浦肯野细胞有树突变短,分枝减少等形态学变化。(4)电镜观察结果:APP/PS1组和3xTg-AD组转基因小鼠均有细胞核凋亡现象,如电子密度增高,核固缩及异染色体边集。两种转基因小鼠小脑皮质内均发现有线粒体肿胀及空泡化,甚至线粒体嵴断裂和结构不完整等现象。同时还伴有细胞骨架破坏,双螺旋细丝样(PHFs)等结构变化。3xTg-AD组甚至还出现微管断裂的现象。此外,APP/PS1组和3xTg-AD组转基因小鼠小脑皮质内平行纤维-浦肯野细胞间突触(PF-PC)的形态学参数也发生明显变化:突触间隙变宽(APP/PS1:P0.001,3xTg-AD:P0.001),突触后致密区(PSD)变薄(APP/PS1:P0.001,3xTg-AD:P0.001),PSD弧度变短(P0.05)及突触曲率变小(P0.001)。结论:(1)AD转基因小鼠小脑内出现AD特异性病理改变。(2)AD小鼠小脑内突触超微结构及相关蛋白发生明显变化,可能与BDNF合成减少有关。(3)APP/PS1组和3xTg-AD组转基因小鼠间,小脑突触超微结构及相关蛋白无显著性差异。
[Abstract]:Objective: This study will use the APPswe/PS1d E9 (APP/PS1) and APPswe/PS1M146V/tau P301L (3xTg-AD) transgenic mice model respectively, using Western-blotting, immunohistochemistry and transmission electron microscopy to observe the expression of synapse related proteins in the cerebellum and ultrastructure changes in the cerebellum of AD model mice, and explore the changes of the AD cerebellum synaptic function. The possible mechanism provides a new theoretical basis for further revealing the role of the cerebellum in the occurrence of AD. Methods: 9 month old, male APP/PS1 double transgenic mice, 3xTg-AD gene mice and control wild C57BL/6J mice (wild type, WT) were divided into wild type group (WT), double rotation genome (APP/PS1), and three transgenic genome (3xTg-AD). It was provided by the genetic engineering platform of the Institute of laboratory animals of the Chinese Academy of Medical Sciences and the The Jackson Laboratory company. The natural circadian rhythms of the feeding environment were illuminated by free feeding and well ventilated. Each group of mice was randomly divided into three groups, which were immunohistochemical, Western-blotting, transmission electron microscopy. (1) immunohistochemistry experiment: The mouse heart was perfused and frozen at -80 C, and then frozen at -80 C, then the brain slices were examined by immunohistochemistry. The thickness of the brain slices was 30 u m, the donkey serum was closed, the first anti 4 centigrade was overnight, and two was hatched, the color, the transparency, and the seal were used for 3 times and 5 min each time. The A beta spots in the cerebellar cortex of the mice were compared with the microscope. The A beta spots in the cerebellar cortex of each group were observed and compared. Under the microscope, the A beta speckles in cerebellar cortex of mice were compared. Under microscope, the A beta speckles in cerebellar cortex of mice were observed and compared. Under microscope, the A beta speckles in cerebellar cortex of mice were compared. Under microscope, the A beta speckles in cerebellar cortex of mice were compared. Under microscope, the A beta spots in cerebellar cortex of mice were compared. Mass (6E10), synaptophysin (Synaptophysin), brain derived neurotrophic factor (Brain-derived neurotrophic factor, BDNF) and its high affinity receptor (Tyrosine kinase B, Trk B). (2) Western-blotting experiment: extraction of mouse cerebellar tissue protein, preparation of gelatin, protein addition, voltage electrophoresis. Then transfer membrane, closed, one anti 4 C over Night, two anti incubation, finally exposed to exposure, using an exposure instrument to analyze the changes in the expression of the strip (Synaptophysin, BDNF, Trk-B and beta -actin). (3) transmission electron microscopy: the cerebellum was removed after the heart perfusion, the cerebellum was cut into the tissue block of 1mm * 1mm x 1mm, and 4% polyformaldehyde was fixed with P H7.2 phosphoric acid buffer solution and placed in osmium. After medium and posterior fixation, double steam rinse, dehydration, acetone and embedding agent, room temperature soaking, epoxy resin embedding, soaking, and polymerization. The ultrathin slice machine section, uranium acetate lead citrate double staining, transmission electron microscope observation and photographing [1], and quantitative analysis of synapse related indexes. Results: (1) cerebellar cortex of group APP/PS1 and 3xTg-AD group transgenic mice A small amount of A beta plaques were deposited. (2) Western-blotting experimental results showed that the content of cerebellar synaptopsin (Synaptophysin) in the APP/PS1 and 3xTg-AD groups decreased significantly (APP/PS1:P0.01,3xTg-AD:P0.01) compared with the WT group (APP/PS1:P0.01,3xTg-AD:P0.01). The immunohistochemical results showed that the cerebellar molecular layer (P0.01) and the Purkinje cell layer (P0.0) of the APP/PS1 group (P0.0). 1) the positive expression of synaptophysin in the granular cell layer (P0.01) was significantly reduced, and the light density in the 3xTg-AD group (P0.01), the Purkinje cell layer (P0.01) and the granular cell layer (P0.01) also decreased significantly, but compared with the APP/PS1 group, there was no statistical difference (P0.05). (3) the two groups of mice in the cerebellum were not significantly changed. (3) Weste The results of rn-blotting test showed that compared with the WT group, the content of BDNF in the cerebellum of APP/PS1 and 3xTg-AD mice decreased significantly (APP/PS1:P0.01,3xTg-AD:P0.01), and the content of Trk-B decreased significantly (APP/PS1:P0.01,3xTg-AD:P0.01). The results of immunohistochemical experiment showed that the molecular layer of the APP/PS1 and 3xTg-AD groups (APP/PS1:P0) (APP/PS1:P0) .01,3xTg-AD:P0.01) and granulosa cell layer (APP/PS1:P0.01,3xTg-AD:P0.01) positive particles were significantly decreased, but the positive expression of BDNF in the molecular layer was not significantly changed (APP/PS1:P0.05,3xTg-AD:P0.05) and Trk-B positive particles in each layer of the cerebellar cortex of 3xTg-AD group and.APP/PS1 group decreased significantly, and the relative light density value was in the molecular layer (APP/PS1:P0.001,3xTg). -AD:P0.001), the Purkinje cell layer (APP/PS1:P0.001,3xTg-AD:P0.001) and the granular cell layer (APP/PS1:P0.001,3xTg-AD:P0.001) were obviously decreased, but there was no statistical difference between the APP/PS1 group and the 3xTg-AD group (APP/PS1:P0.05,3xTg-AD:P0.05). In addition, the morphology of the Purkinje cells in the AD mice was shortened and the branching decreased. Changes. (4) the results of electron microscopy: nuclear apoptosis in APP/PS1 and 3xTg-AD transgenic mice, such as increased electron density, nuclear pyknosis and heterosomal edge set. Mitochondria swelling and vacuolization were found in the cerebellar cortex of the two transgenic mice, even mitochondrial crista fracture and incomplete structure. Skeleton destruction, double spiral filament like (PHFs) and other structural changes in the.3xTg-AD group even appeared microtubule fracture. In addition, the morphological parameters of the parallel fiber - Purkinje intercellular synapse (PF-PC) in the cerebellar cortex of APP/PS1 and 3xTg-AD transgenic mice also changed obviously: the synaptic gap widened (APP/PS1:P0.001,3xTg-AD:P0.001). The post touch dense region (PSD) thinning (APP/PS1:P0.001,3xTg-AD:P0.001), PSD radians shortened (P0.05) and the synaptic curvature decreased (P0.001). Conclusion: (1) there are AD specific pathological changes in the cerebellum of (1) AD transgenic mice. (2) the ultrastructure of the synapse in the cerebellum and the significant changes in the associated egg white hair in the cerebellum of AD mice may be related to the decrease of BDNF synthesis. (3) APP/PS1 and 3xT There was no significant difference in the ultrastructure of cerebellar synapses and related proteins between transgenic mice in group g-AD.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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