摘要
目的:神经系统常见的神经退行性疾病如阿尔茨海默病(Alzheimer’s Disease,AD)、帕金森病(Parkinson’s Disease,PD)、亨廷顿病(Huntington’s Disease,HD)和肌萎缩侧索硬化(Amyotrophic lateral sclerosis,ALS)等病因大多不明,发病机制和病理表现复杂,临床表现各异。但此类疾病的共同特点是中老年人发病,其发病率随年龄的增加而增高;而且共同的病理特征为在特异性神经元中存在异常蛋白聚集体和/或包涵体,并且这些异常蛋白聚集体或包涵体呈泛素阳性反应。因此,了解泛素在人类老化过程中的表达特征无疑对研究神经变性疾病有重要的意义。
泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)是细胞内蛋白质降解的主要途径,降解80%以上的细胞内错误折叠蛋白或其他突变蛋白。研究认为泛素蛋白酶体降解通路失调可导致异常蛋白在细胞内积聚,并进一步引起细胞功能紊乱及变性。UPS功能异常可以发生于UPS途径的任何一个环节,如泛素化过程中泛素量的失衡,泛素结合过程中泛素活化酶E1,泛素结合酶E2,泛素连接酶E3功能失调,蛋白酶体降解水平失调或去泛素化水平失调等。观察泛素蛋白酶体降解系统在神经退行性疾病易感组织及不同年龄阶段的表达具有重要的意义。以往对于泛素蛋白表达与年龄相关性的研究较少,国内尚无此类研究。因此本实验旨在观察泛素蛋白在中枢神经退行变性疾病易感区及不同年龄阶段的表达及分布特点,探讨泛素蛋白水平与神经变性疾病相关性,从而为理解神经变性疾病的年龄相关性提供理论依据。
方法:年龄分别为3个月、6个月、9个月、12个月的正常健康ICR小鼠各3只,雌雄不限,10%水合氯醛350mg/kg腹腔注射麻醉后,心脏灌注4%多聚甲醛固定20min,分别冠状切取嗅球后至视交叉前脑组织、冠状切取视交叉后1mm至中脑下丘之前的脑组织、切取脊髓腰膨大及部分肝脏组织(作为对照组织),均以4%多聚甲醛固定48h,梯度酒精脱水,二甲苯透明,石蜡包埋,常规组织切片(5μm厚)。5μm厚的石蜡切片用于免疫组织化学方法检测泛素蛋白的表达及分布特点。
采用真彩色医学图像分析软件(Midia Cybernet公司Image-pro Plus5.0图像分析系统)和人工计数方法对小鼠大脑运动皮层Betz大锥体细胞层、海马CA1区锥体细胞层、中脑黒质致密部、腰膨大脊髓前角及肝脏组织进行图像采集、光密度分析和泛素蛋白阳性细胞计数。
结果:
1泛素蛋白阳性区域平均积分光密度(MIOD)
大脑运动皮层Betz大锥体细胞层、海马CA1区锥体细胞层、中脑黒质致密部、肝脏组织泛素蛋白阳性区域的平均积分光密度均未随增龄而有明显变化(P>0.05);脊髓前角泛素蛋白阳性区域平均积分光密度随增龄存在统计学差异(P<0.05),其中6个月月龄组与3个月月龄组相比,平均积分光密度明显降低(P<0.05),9个月月龄组与6个月月龄组相比,平均积分光密度亦有明显降低(P<0.05),12个月月龄组与9个月月龄组相比,平均积分光密度无差别(P>0.05)。
2泛素蛋白阳性细胞计数
2.1大脑运动皮层Betz大锥体细胞层3个月月龄组、6个月月龄组、9个月月龄组、12个月月龄组相邻组之间相比,泛素蛋白阳性神经元数目均无明显变化(P>0.05);而12个月月龄组与6个月月龄组相比,与3个月月龄组相比,泛素蛋白阳性神经元数目明显减少(P<0.05);9个月月龄组与3个月月龄组相比泛素蛋白阳性神经元数目亦明显减少(P<0.05)。
2.2海马CA1区锥体细胞层6个月月龄组与3个月月龄组相比泛素蛋白阳性神经元数目无明显变化(P>0.05);6个月、9个月、12个月三个月龄组之间相比泛素蛋白阳性神经元数目明显减少(P<0.05)。
2.3中脑黒质致密部6个月月龄组与3个月月龄组相比泛素蛋白阳性神经元数目无明显变化(P>0.05);9个月月龄组与6个月月龄组相比,与3个月月龄组相比泛素蛋白阳性神经元数目均有明显减少(P<0.05);12个月月龄组与6个月月龄组相比,与3个月月龄组相比泛素蛋白阳性神经元数目均有明显减少(P<0.05)。2.4腰膨大脊髓前角9个月、12个月月龄组分别与3个月月龄组相比泛素蛋白阳性神经元数目均有明显减少(P<0.05);9个月、12个月月龄组分别与6个月月龄组相比,泛素蛋白阳性神经元数目均有明显减少(P<0.05),12个月月龄组与9个月月龄组相比,泛素蛋白阳性神经元数目无明显变化(P>0.05)。
2.5肝脏组织各月龄组泛素蛋白阳性细胞数未见明显变化(P>0.05)。
结论:随着年龄增长,大脑运动皮层、海马、黒质及腰膨大脊髓前角神经退行性疾病易感区内泛素蛋白表达阳性神经元数目逐渐减少;而除了腰膨大脊髓前角,泛素蛋白表达在神经元内含量均不随年龄增长而减少。
Objective: Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS) are very common in clinic, most of which are unclear in etiology, complex in pathogenesis and pathology, and vary in clinical manifestations. However, there are two common features of these diseases. The onset stage of these diseases is in old age and its incidence increases with aging; The common pathological hallmark of many neurodegenerative diseases is the presence of ubiquitin-positive, intra or extracellular inclusion bodies in affected regions of the brain. So people pay close attention to the role of the ubiquitin-proteasome system in the neurodegenerative diseases.
Ubiquitin-proteasome system (UPS) is the main path of the protein degradation within cells, which can degrade 80% of the intracellular misfolded protein or mutein. Previous studies suggested that disorders of ubiquitin-proteasome degradation pathway can lead to the accumulation of abnormal proteins in cells, leading to cell dysfunction and degeneration. UPS dysfunction can occur in any part of UPS pathway, such as the imbalance in the quantity of ubiquitin, dysfunction of E1, E2 or E3 in ubiquitin-binding process, disorder of proteasome degradation or deubiquitination. Therefore, it is significant to observe the expression of ubiquitin-proteasome system in the areas of liability to neurodegenerative diseases in different age stages.
The study for the correlation of ubiquitin protein expression and age is less in the past, and there is no such reaserch in this field in our country. Therefore, this experiment aims to observe the expression and distribution features of the ubiquitin protein in susceptible areas of neurodegenerative diseases in different age stages, approach the correlation of the level of ubiquitin protein and neurodegenerative diseases and thus provide a theoretical basis for understanding age-relateness of neurodegenerative diseases.
Methods: 3 healthy ICR mice were used in each group of aged 3 months, 6 months, 9 months and 12 months. After anesthesia, animal tissues were fixated via heart perfusion by 4% paraformaldehyde for 20min. Brain tissue from posterior of olfactory bulb to anterior of optic chiasma, brain tissue from 1mm after optic chiasma to midbrain inferior colliculus were cut coronarily; intumescentia lumbalisa of spinal cord and a part of liver tissue(as control group tissue) were cut. All the tissues cut above were fixated in 4% paraformaldehyde for 48h. Then they were dehydrated by gradient ethanol, transparentized by xylene, embedded by paraffin and made into conventional histological sections futher (5μm thick). 5μm thick paraffin sections were used to Immunohistostain for detection of ubiquitin-protein expression and distribution.
True color medical image analysis software (Midia Cybernetic Corpo- ration Image-pro Plus5.0 image analysis system) and manual counting methods were used to capture images from Betz's cell layer of cerebral motor cortex, hippocampal CA1 pyramidal cell layer, the pars compacta of midbrain nigral, spinal cord anterior horn of lumbar intumescentia and the liver tissue, to analysis optical density and to counts ubiquitin-positive cell.
Results:
1 Mean integral opitcal density (MIOD) of Ubiquitin-positive staining region
The MIOD values of the ubiquitin-positive region of Betz's cell layer of cerebral motor cortex, hippocampal CA1 pyramidal cell layer, the pars compacta of midbrain nigral and liver tissue showed that there was no significant differencese (P>0.05). The MIOD values of ubiquitin-positive region in spinal cord anterior horn of lumbar intumescentia were decreased with aging (P<0.05). The MIOD value of the 12 months old group was significantly lower than that of 3 months old group (P<0.05), 6 months old group and 9 months old group. 9 months old group was also significantly lower than the 3 months old group (P<0.05).
2 Count of ubiquitin-positive cell
2.1 The Betz's cell layer of cerebral motor cortex
There was no significant diference in number of ubiquitin-positive cell between the adjacent groups of 3 months old group, 6 months old group, 9 months old group and 12 months old group (P>0.05); 12 months old group was lower than 6 months old group and 3 months old group (P<0.05). 9 months old group was lower than 3 months old group (P<0.05).
2.2 The pyramidal cell layer of hippocampal CA1 subfield
Comparisons showed significantly and gradually decrease in the number of ubiquitin-positive cell among 3 months old group, 6 months group, 9 months group and 12 months group (P<0.05).
2.3 The pars compacta of midbrain nigral
There was no significant diference in number of ubiquitin-positive cell between 6 months old group and 3 months old group (P>0.05). 9 months old group was significantly lower in the number of ubiquitin-positive cell compared with both 6 months old group (P<0.05) and 3 months old group (P<0.05). 12 months old group was significantly lower in the number of ubiquitin-positive cell compared with both 6 months old group (P<0.05) and 3 months old group (P<0.05).
2.4 Spinal cord anterior horn of lumbar intumescentia
There was no significant diference in number of ubiquitin-positive cell between 6 months old group and 3 months old group (P>0.05). 9 months old group was significantly lower in the number of ubiquitin-positive cell compared with both 6 months old group (P<0.05) and 3 months old group (P<0.05). 12 months old group was significantly lower in the number of ubiquitin-positive cell compared with both 6 months old group (P<0.05) and 3 months old group (P<0.05).
2.5 Liver tissue
The ubiquitin-positive cell number of each month age group showed no significant difference (P>0.05).
Conclusions:
The number of ubiquitin-positive cell in the areas of liability to neurodegenerative diseases, such as cerebral motor cortex, hippocampus, midbrain nigral and spinal cord anterior horn of lumbar intumescentia, decreased with aging. The MOID of reservated ubiquitin-positive neuron has no significant difference except the spinal cord anterior horn of lumbar intumescentia.
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