小鼠老年痴呆模型的建立及甘丙肽的表达研究

发布时间：2018-03-10 11:55

本文选题：老年性痴呆　切入点：学习记忆　出处：《华东师范大学》2007年硕士论文　论文类型：学位论文

【摘要】： [目的] 运用生化、组织、行为学方法评价D-半乳糖引发老年性痴呆(Alzheimer’s Disease，，AD)小鼠模型，测定海马脑区甘丙肽及其受体表达的变化；探讨甘丙肽在AD发生过程中可能的作用及其机制。 [方法] 选用20g左右的BL/C57小鼠50只，分为模型组和对照组，各25只。模型组腹腔注射D-半乳糖150mg/kg，每天一次，连续90d，制备老年痴呆动物模型。对照组注射等量的0.9％的生理盐水。通过Y-型迷宫比较两组小鼠的学习记忆能力，第一天检测小鼠学习能力，间隔24小时后检测小鼠记忆能力。行为学实验结束后，每组取10只小鼠，测定蛋白质含量和AChE、ChAT、MAO、MDA、GSH-PX、SOD、Na~+K~+-ATP酶，按试剂盒说明书所述步骤操作。各组小鼠腹腔注射戊巴比妥钠60mg/kg麻醉，用多聚甲醛进行心脏灌流，，取脑感觉运动区皮层，置于戊二醛中，透射电镜观察。荧光显微镜配合免疫组织化学观察小鼠脑的矢状冷冻切片海马区域甘丙肽的表达，荧光显微镜检测照相。 Realtime-PCR检测小鼠脑区前额叶、中脑导水管周围灰质中甘丙肽及其受体1、2、3表达状况。 [结果] (1)行为学检测结果说明模型小鼠的学习记忆能力明显低于对照小鼠。 (2)生化指标检测出模型组AChE、ChAT、GSH-PX、SOD、Na~+K~+-ATP酶活性明显降低，MAO活性上升，MDA含量明显上升。 (3)电镜病理切片显示模型组的细胞核明显萎缩；线粒体嵴消失，或产生空泡。对照组细胞核正常，线粒体嵴清晰。 (4)免疫荧光检测海马区域甘丙肽含量表达差异，模型组甘丙肽表达明显高于对照组。 (5) Realtime-PCR检测出模型组前额叶、中脑导水管周围灰质区域甘丙肽及其受体1、2、3的mRNA含量高于对照组。 [结论] (1)模型建立比较成功，为实验提供可靠的前提。 (2)老年痴呆小鼠氧化还原酶SOD、GSH-PX、AChE、ChAT、Na~+-K~+-ATP酶活性下降，MDA含量上升、MAO活性上升，说明体内氧自由基含量明显升高，符合衰老的特征。 (3)痴呆小鼠的脑细胞发生了病理变化：细胞核萎缩，线粒体破裂或呈空泡。 (4)甘丙肽在老年痴呆小鼠海马中呈阳性增加表达效应。 (5)前额叶、中脑导水管周围灰质脑区甘丙肽受体出现高表达。
[Abstract]:[purpose]. Methods of biochemistry, tissue and behavior were used to evaluate the model of Alzheimer's dementia induced by D-galactose-induced Alzheimer's Disease (AD), to determine the changes of glycine and its receptor expression in hippocampus, and to explore the possible role and mechanism of galanin in the pathogenesis of AD. [methods]. Fifty BL/C57 mice with 20 g or so were divided into two groups: model group and control group. The model group was injected intraperitoneally with D-galactose 150 mg / kg, once a day for 90 days, and the control group was injected with the same amount of 0.9% normal saline. The learning and memory abilities of the two groups were compared by Y- maze. The learning ability of the mice was measured on the first day and the memory ability of the mice was measured after 24 hours interval. At the end of behavioral experiment, 10 mice in each group were selected. The protein content and the protein content in each group were determined, and the activity of GSH-PXX SODN Na ~ K ~ -ATPase was determined according to the procedure described in the specification of the kit. The mice were anesthetized with 60 mg / kg pentobarbital sodium intraperitoneally and perfused with paraformaldehyde. The cortex of sensorimotor area was taken and placed in glutaraldehyde and observed by transmission electron microscope. The expression of galanin in hippocampus of sagittal frozen section of mouse brain was observed by fluorescence microscope and immunohistochemistry. Realtime-PCR was used to detect the expression of glycidyl peptide and its receptor 1 / 2 in the prefrontal lobe and periaqueductal gray of mice. [results]. The results of behavioral examination showed that the learning and memory ability of the model mice was significantly lower than that of the control mice. In the model group, the activity of GSH-PXX SODN Na ~ K ~ -ATPase decreased obviously and the content of MDA increased obviously. Electron microscopic pathological sections showed that the nuclei of the model group were obviously atrophied, the mitochondrial crest disappeared or vacuoles were produced, and the nuclei of the control group were normal and the mitochondrial crest was clear. (4) Immunofluorescence assay showed that the level of galanin in the model group was significantly higher than that in the control group. (5) the content of mRNA in prefrontal lobe and periaqueductal gray matter area of model group was higher than that in control group by Realtime-PCR. [conclusion]. 1) the establishment of the model is successful and provides a reliable premise for the experiment. (2) in senile dementia mice, the activity of redox enzyme SODH-PXH- AChEATE (Na ~ -K ~ -ATPase) decreased and the activity of Mao increased, indicating that the content of oxygen free radicals in the body was obviously increased, which was consistent with the characteristics of senility. 3) pathological changes of brain cells in dementia mice: nucleus atrophy, mitochondria rupture or vacuole. 4) the expression of glycidyl in hippocampus of senile dementia mice was increased. The prefrontal lobe and periaqueductal gray area of the midbrain showed high expression of glycine receptor.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332

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