阿尔茨海默病小鼠大脑和脊髓自噬变化的研究

目的 研究阿尔茨海默病(AD)小鼠大脑和脊髓自噬变化情况.方法 分离3月、8月和11月龄AD小鼠和同龄正常对照小鼠(n=6)的大脑和脊髓,左侧半脑和脊髓腰段用于组织切片的制备,右侧半脑及剩余脊髓用于提取蛋白.采用免疫组织化学方法观察脑内和脊髓淀粉样斑块；激光共聚焦显微镜观察自噬指标LC3在脑内和脊髓运动神经元中的分布情况；Westernblotting检测8月龄小鼠大脑及脊髓LC3-Ⅱ和Beclin 1蛋白表达水平；Nissl染色计数脊髓运动神经元.结果 免疫组织化学检测结果显示:3月龄AD小鼠脑内无明显淀粉样斑块沉积,8月龄时皮层和海马出现大量斑块,11月龄时斑块数量明显增多；AD小鼠脊髓腰段运动神经元内存在淀粉样蛋白,但未检测到细胞外斑块沉积.激光共聚焦显微镜观察结果显示；8月龄AD小鼠大脑皮层LC3阳性自噬小泡数量多于对照小鼠；3月龄AD小鼠脊髓运动神经元内存在LC3阳性自噬小泡,自噬小泡数量在8月龄和11月龄时明显增多.Western blotting结果显示:与8月龄对照小鼠比较,8月龄AD小鼠脑组织LC3-Ⅱ蛋白表达升高(P＜0.01),脑组织Beclin 1 蛋白表达降低(P＜0.05),脊髓LC3-Ⅱ蛋白表达升高(P＜0.05),脊髓Beclin 1蛋白表达水平无明显变化(P＞0.05).Nissl染色结果显示:与8月龄对照小鼠比较,8月龄AD小鼠脊髓腰段运动神经元数量无明显变化(P＞0.05).结论 AD小鼠大脑和脊髓存在自噬改变,自噬水平的变化可能与细胞内淀粉样蛋白有关.

Autophagic changes in brain and spinal cord of mice with Alzheimer's disease

Objective To investigate the autophagic changes in the brain and spinal cord of mice with Alzheimer’s disease(AD).Methods The brain and spinal cord of AD mice aged 3 months,8 months and 11 months and normal controls(n=6) of the corresponding ages were isolated.The left side of brain and the lumbar segment of spinal cord were used for histological sections,and the right side of brain and the remaining part of spinal cord were used for protein extraction.Immunohistochemistry was employed to detect the amyloid plaque in the brain and spinal cord,confocal microscopy was performed to observe the distribution of LC3-positive autophagic vesicles in the brain and motor neuron in the spinal cord,the expression of LC3-II and Beclin 1 protein in the brain and spinal cord of mice aged 8 months was determined by Western blotting,and Nissl staining was adopted to quantify the motor neuron in the spinal cord.Results The immunohistochemistry revealed that there was no amyloid plaque in the brain of AD mice aged 3 months,a large number of plaques emerged in the cortex and hippocampus in mice aged 8 months,and the quantity of plaques increased significantly in mice aged 11 months.There was intraneuronal amyloid in the lumbar spinal cord of AD mice,while no extracellular plaque was detected.The confocal microscopy revealed that the quantity of LC3-positive autophagic vesicles in the brain cortex of AD mice aged 8 months was significantly larger than that in control group,there were LC3-positive autophagic vesicles in motor neuron in the spinal cord of AD mice aged 3 months,and the quantities of autophagic vesicles significantly increased in mice aged 8 months and 11 months.Western blotting indicated that compared with the corresponding controls,the expression of LC3-II protein in brain tissues of AD mice aged 8 months was significantly higher(P<0.01),the expression of Beclin 1 protein in brain tissues was significantly lower(P<0.05),the expression of LC3-II protein in spinal cord was significantly higher(P<0.05),and the expression of Beclin 1 protein in spinal cord was not significantly changed(P>0.05).Nissl staining demonstrated that there was no significant difference in the quantity of motor neuron of lumbar segment of spinal cord between AD mice aged 8 months and corresponding controls(P>0.05).Conclusion There are significant autophagic changes in the brain and spinal cord of AD mice,and the change of autophagy may be associated with the change of intraneuronal amyloid levels.