G蛋白调节子Rgs5在炎症调控中作用的研究

发布时间：2018-01-02 05:30

  本文关键词：G蛋白调节子Rgs5在炎症调控中作用的研究　出处：《南京医科大学》2013年硕士论文　论文类型：学位论文

  更多相关文章： G蛋白调节子Rgs5 星形胶质细胞 神经炎症 多巴胺能神经元 帕金森病

【摘要】：帕金森病（Parkinson's disease，PD）是仅次于阿尔茨海默病（Alzheimer'sdisease，AD）之后的第二大神经退行性疾病，其主要病理表现是中脑黑质多巴胺神经元的选择性损伤。目前研究发现，其可能的致病机制包括α-synuclein的聚集、氧化应激反应、线粒体复合物功能的衰退、泛素蛋白酶系统功能障碍等。近年来，通过流行病学、神经遗传学和临床神经病理学研究发现，慢性神经炎症随着大脑衰老不断增强，而在包括PD在内的多种神经退行性疾病中，神经炎症异常增加，并可能在退行性病变的发生和发展过程中发挥一定作用。 中枢神经系统中，调节炎症反应的分子机制并不明确。在本研究中，我们探讨了G蛋白偶联信号通路调节子Regulators of G protein signaling5(Rgs5)在神经炎症中可能的作用。我们发现，Rgs5可能参与脑内星形胶质细胞对大脑免疫炎症过程的调节。在生理情况下，Rgs5敲除小鼠中促炎症因子IL-1β的蛋白水平上调，在MPTP诱导的小鼠PD模型中，中脑多巴胺神经元更易受损。体外培养的Rgs5敲除的星形胶质细胞在促炎症因子TNF-α刺激下，IL-1β和TNF-α蛋白水平和mRNA的水平都显著上调。这些结果表明，星形胶质细胞中的Rgs5可能在调节神经炎症过程中发挥一定作用，这对进一步理解星形胶质细胞在脑内炎症反应中的作用有一定意义。 目的：在整体、细胞及分子水平研究Rgs5对PD中多巴胺神经元的选择性损伤和神经炎症的影响。 方法：1.利用野生型、Rgs5基因敲除小鼠、条件敲除小鼠结合MPTP诱导的帕金森病小鼠模型，进行多种整体动物的研究；2.应用免疫组织化学法研究Rgs5敲除小鼠中多巴胺能神经元损伤、星形胶质细胞及小胶质细胞活化情况；3.原代培养小鼠中脑多巴胺神经元，给予MPP+神经毒素刺激，检测神经元中Rgs5对神经元存活的影响。4.原代培养全脑星形胶质细胞，给予TNF-α和条件培养基处理，应用Western Blotting和qPCR检测胶质细胞中Rgs5对炎症因子的调节作用。 结果： 1. Rgs5敲除后，生理条件下，纹状体组织样品中蛋白水平炎症因子IL-1β有上调，，但炎症因子IL-1β、IL-12β、TNF-mRNA与对照组无显著差异。在体外培养的Rgs5敲除星形胶质细胞中，IL-1β的蛋白水平上调，但其mRNA水平同样无显著变化。 2.在MTPT诱导的帕金森病模型小鼠中，Rgs5敲除小鼠较野生型小鼠黑质多巴胺神经元损伤更加严重。 3.体外培养小鼠中脑多巴胺神经元，模拟在体的MPTP模型给予MPP+刺激，Rgs5敲除与野生型小鼠多巴胺神经元的死亡率无显著差异。 4.原代培养Rgs5敲除的星形胶质细胞，给予TNF-α刺激后，IL-1β的蛋白和mRNA水平进一步上调。而MPTP急性PD模型中，星形胶质细胞中条件敲除Rgs5小鼠并未引起黑质多巴胺神经元的进一步损伤。 结论：Rgs5基因敲除使得脑内基础炎症水平增强，且使中脑黑质多巴胺神经元对神经毒素MPTP更加易感。 本文工作的创新之处在于： 1.发现在脑内Rgs5与神经炎症存在一定关联；Rgs5可能参与调控PD模型中中脑黑质多巴胺神经元的存活。 2.探讨了星形胶质细胞中的Rgs5对神经炎症可能的贡献。
[Abstract]:Parkinson's disease (Parkinson's disease PD) is after Alzheimer's disease (Alzheimer'sdisease, AD) second major neurodegenerative disease after, its main pathological manifestation is selective injury of dopaminergic neurons in substantia nigra. The current study found that the possible pathogenic mechanism including alpha -synuclein aggregation, oxidative stress, mitochondrial complex function decline the ubiquitin proteasome system dysfunction. In recent years, the epidemiology, genetics and clinical study found that nerve neuropathology, chronic inflammation with brain aging increasing, and in a variety of neurodegenerative diseases including PD, abnormal increase of neuroinflammation, and may play a role in the occurrence and development of degenerative diseases.
In the central nervous system, the molecular mechanisms that regulate the inflammatory response is not clear. In this study, we investigate the G protein coupled signal pathway in regulation of Regulators of G protein signaling5 (Rgs5) in the role of neuroinflammation. We found that Rgs5 may be involved in the regulation of glial cells in the brain star on the brain immune inflammatory process in physiological conditions, Rgs5 knockout mice proinflammatory factor IL-1 beta protein levels increased in mouse PD model induced by MPTP in midbrain dopamine neurons are easy to be damaged. In vitro Rgs5 knockout astrocyte cells in cytokine stimulated with TNF-, IL-1 beta and TNF- protein level and the level of mRNA was significantly upregulated. These results suggest that Rgs5 in astrocytes may play a role in the regulation of inflammatory process, the further understanding of astrocytes in brain inflammatory reaction The role of the should be of certain significance.
Objective: To study the effects of Rgs5 on selective injury and neuroinflammation of dopamine neurons in PD, as a whole, cell and molecular level.
Methods: 1. using wild type Rgs5 gene knockout mice, conditional knockout mice with Parkinson's disease mouse model induced by MPTP, a variety of animal research; research of Rgs5 2. by immunohistochemistry in the knockout mice injury of dopaminergic neurons, glial cells and microglia activation of midbrain dopamine in mice; 3. primary cultured neurons, MPP+ neurons in neurotoxin stimulation, detection effect of Rgs5 on neuronal survival in.4. primary cultured cerebral astrocytes, TNF- alpha and conditioned medium, regulation of Blotting and application of Western qPCR detection in Rgs5 glial cells on inflammatory cytokines.
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