中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (2): 224-229.doi: 10.3969/j.issn.2095-4344.2016.02.013

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

β-淀粉样蛋白25-35致伤对PC12神经元突触相关蛋白表达的影响

张 爽1，黄昕艳2，刘 爽3，李艳君3，赵锦程3   

  1. 佳木斯大学，1药学院，3基础医学院，黑龙江省佳木斯市 154007；2佳木斯大学附属第二医院，黑龙江省佳木斯市 154007
  • 出版日期:2016-01-08 发布日期:2016-01-08
  • 通讯作者: 赵锦程，教授，佳木斯大学基础医学院，黑龙江省佳木斯市 154007
  • 作者简介:张爽，女，1989年生，汉族，在读硕士，主要从事神经退行性变机制研究。
  • 基金资助:
    黑龙江省教育厅科研项目(1251666)；佳木斯大学基础研究重点项目(12Z1201502)；佳木斯大学研究生科技创新项目(LZZ2014_030)

Effects of amyloid-beta 25-35 on expression of synapse-associated proteins in PC12 neurons

Zhang Shuang1, Huang Xin-yan2, Liu Shuang3, Li Yan-jun3, Zhao Jin-cheng3   

  1. 1College of Pharmacy, Jiamusi University, Jiamusi 154007, Heilongjiang Province, China; 2Second Affiliated Hospital of Jiamusi University, Jiamusi 154007, Heilongjiang Province, China; 3Basic Medical College, Jiamusi University, Jiamusi 154007, Heilongjiang Province, China
  • Online:2016-01-08 Published:2016-01-08
  • Contact: Zhao Jin-cheng, Professor, Basic Medical College of Jiamusi University, Jiamusi 154007, Heilongjiang Province, China
  • About author:Zhang Shuang, Studying for master’s degree, College of Pharmacy, Jiamusi University, Jiamusi 154007, Heilongjiang Province, China
  • Supported by:

    the Scientific Research Project of Heilongjiang Educational Department, No. 1251666; the Basic Research Project of Jiamusi University, No. 12Z1201502; the Graduate Scientific Innovation Project of Jiamusi University, No. LZZ2014_030

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摘要：

文章快速阅读：

文题释义：

β淀粉样蛋白：是由淀粉样前体蛋白经β-和γ-分泌酶的蛋白水解作用而产生的含有39-43个氨基酸的多肽。它可由多种细胞产生，循环于血液、脑脊液和脑间质液中，大多与伴侣蛋白分子结合，少数以游离状态存在。人体内β淀粉样蛋白最常见的亚型是β淀粉样蛋白1-40和 β淀粉样蛋白1-42。在人脑脊液和血中，β淀粉样蛋白1-40分别比β淀粉样蛋白1- 42的含量水平高10 倍和1.5倍，β淀粉样蛋白1-42具有更强的毒性，且更容易聚集，从而形成β淀粉样蛋白沉淀的核心，引发神经毒性作用。

突触：是一个神经元的冲动传到另一个神经元或传到另一细胞间的相互接触的结构。突触是神经元之间在功能上发生联系的部位，也是信息传递的关键部位。在光学显微镜下观察，可以看到一个神经元的轴突末梢经过多次分支，最后每一小支的末端膨大呈杯状或球状，叫做突触小体。这些突触小体可以与多个神经元的细胞体或树突相接触，形成突触。从电子显微镜下观察，可以看到，这种突触是由突触前膜、突触间隙和突触后膜3部分构成。

 

背景：脑内β-淀粉样蛋白的聚集可诱导神经细胞凋亡，大量神经元及突触的缺失和功能的损害尚无有效的干预手段，提高突触可塑性为治疗早期阿尔茨海默病提供重要方向。
目的：筛选最佳的阿尔茨海默病模型，检测β-淀粉样蛋白25-35致伤PC12神经元的突触相关蛋白表达。
方法：采用50 μg/L神经生长因子诱导PC12细胞分化为神经元样细胞，以不同浓度β-淀粉样蛋白25-35致伤PC12神经元样细胞。应用CCK8法检测细胞生存率。神经颗粒素、神经调节素免疫荧光染色观察模型细胞的形态学变化，Western blot法检测神经颗粒素、CAMKⅡ、PSD-95蛋白表达水平。
结果与结论：随着β-淀粉样蛋白25-35浓度增高和作用时间的延长，PC12神经元生存率呈剂量依赖性降低；可见突触长度变短、神经元萎缩、神经元彼此连接疏松；神经颗粒素、CAMKⅡ、PSD-95蛋白表达均下调。结果提示，10 μmol/L β-淀粉样蛋白25-35、48 h是筛选早期PC12神经元阿尔茨海默病细胞模型的最佳干预浓度和时间。 
ORCID: 0000-0002-7563-0184(赵锦程)

关键词: 组织构建, 组织工程, PC12细胞株, β-淀粉样蛋白, 神经元, 阿尔茨海默病, 突触后致密物, 突触可塑性

Abstract:

BACKGROUND: An amyloid-beta (Aβ) aggregation in the brain can induce nerve cell apoptosis, loss of synapses and functional damage. However, there is still no effective intervention. Improving the synaptic plasticity provides an important direction for the treatment of early Alzheimer’s disease.
OBJECTIVE: To screen the best model of Alzheimer’s disease and to explore the expression of synapse-associated proteins in Aβ25-35-injured PC12 neurons.
METHODS: PC12 cells were induced by 50 μg/L nerve growth factor to differentiate into neuronal-like cells. Then, these cells were treated with Aβ25-35 at different concentrations. Consequently, cell survival rate was detected using cell counting kit-8; neurogranin and neuregulin immunofluorescence stainings were used to observe morphological changes of model cells; western blot used to detect the expression level of neurogranin, calmodulin kinase II, postsynaptic density-95 proteins.
RESULTS AND CONCLUSION: Over time, the survival rate of PC12 neurons induced by Aβ25-35 was decreased in a dose-dependent manner. Shortened synaptic length, neuronal atrophy and sparsely interconnected neurons were visible. Expression levels of neurogranin, calmodulin kinase II and postsynaptic density-95 proteins were all down-regulated. These findings indicate that to screen the cell model of Alzheimer’s disease, the optimal concentration and interventional time of Aβ25-35 are 10 μmol/L and 48 hours, respectively.