IFN-γ介导T细胞参与炎性痛慢性化机制的研究
发布时间：2018-09-06 10:17
【摘要】：研究目的病理性疼痛(又称为慢性痛)已经成为全球最大疾病负担,炎性痛是临床病理性疼痛的主要类型之一。既往研究发现,在组织损伤或炎症发生后,脊髓小胶质细胞和星形胶质细胞合成、释放多种促炎细胞因子、趋化因子和神经营养因子等,彼此相互影响,构成局部炎症微环境,最终增强兴奋性突触传递,即中枢敏化的形成,机体出现痛觉过敏和痛觉异常,但中枢敏化的长期维持或疼痛慢性化的关键机制仍不清楚。神经系统退行性疾病多由自身抗原特异性地T细胞介导,且常常伴有疼痛症状,如分泌IFN-γ的Th1细胞和分泌IL-17的Th17细胞均参与多发性硬化的病理过程。T细胞对神经病理性疼痛的影响逐渐被发现,目前认为其作用与神经病理性疼痛的慢性化相关,至于具体的T细胞亚型,现有证据趋向于Th1细胞,Th17细胞还有待进一步研究。T细胞调控疼痛的具体分子作用机制,以及它们对炎性痛的发生、发展是否有影响及作用机制目前研究较少。外周血T细胞参与脊髓对病理性疼痛的调控,就必须向脊髓中枢募集,需要通过血-中枢神经系统屏障进入脊髓。炎性损伤发生后,脊髓胶质细胞激活产生的多种炎症因子如TNF-α、IL-1β、CCL2等可能会增加血-中枢神经系统屏障的通透性,提高对外周免疫细胞的趋化作用,其中星形胶质细胞更是参与了血-中枢神经系统屏障的构成。此外有研究发现小胶质细胞和星形胶质细胞在炎症等适宜刺激下可上调表达MHC-Ⅱ类分子,可能是中枢神经系统的抗原递呈细胞。据此我们推测胶质细胞的活化尤其是星形胶质细胞的活化可能为T细胞浸润至脊髓发挥免疫效应、维持病理性疼痛提供了必要条件。研究方法为验证以上推测,本课题利用单关节炎大鼠炎性痛模型和原代星形胶质细胞,运用免疫荧光组织化学方法、流式细胞术、蛋白质免疫印迹等实验方法,依以下步骤初步探讨T细胞对炎性痛的贡献、分子机制,以及胶质细胞对它的调节作用。一、观察单关节炎大鼠脊髓腰膨大中T细胞数量及胶质细胞活化情况建立单关节炎大鼠(monoarthritis rats,MA)炎性痛模型,分离MA组和假手术组大鼠致炎后第7天、10天的脊髓腰膨大,检测T细胞数量、分布位置及活化情况,同时检测小胶质细胞离子型钙结合受体分子-1(Ionized calcium-binding adaptor molecule-1,Iba1)和星形胶质细胞胶质纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)的表达水平,观察与比较T细胞数量与胶质细胞活化的动态变化。二、分析探讨脊髓胶质细胞的活化状态与脊髓T细胞数量及活性的关系建立MA模型,通过药理学手段抑制小胶质细胞和星形胶质细胞的活化,检测PWT,以及脊髓腰膨大中T细胞数量及IFN-γ的表达水平,初步探讨胶质细胞活化对T细胞的调节作用。三、分析探讨炎性痛大鼠脊髓IFN-γ表达与星形胶质细胞活化的相关性通过药理学手段改变MA及naive组大鼠脊髓IFN-γ的表达水平后检测机械缩腿反应阈值(Paw withdrawal threshold,PWT),以及星形胶质细胞GFAP的表达水平。并进一步检测经IFN-γ孵育后的原代星形胶质细胞中NF-κBp65的磷酸化水平,明确IFN-γ对星形胶质细胞的调节作用。结果一、单关节炎大鼠脊髓腰膨大中T细胞数量及胶质细胞活化情况致炎后脊髓T细胞浸润及胶质细胞活化:脊髓腰膨大中的T细胞数目在致炎后第7天、10天明显增加,并且在第10天增加更显著。T细胞主要分布在脊髓背角。IFN-γ主要与T细胞标记CD3共标,而Th17标志性细胞因子IL-17主要与星形胶质细胞标记GFAP共标。在术后第7天和第10天,小胶质细胞Iba1和星形胶质细胞GFAP表达水平均上调,其中Iba1和GFAP分别在第7天、10天上调更显著。二、脊髓胶质细胞的活化状态与脊髓T细胞数量及活性的关系星形胶质细胞调节脊髓T细胞相关因子IFN-γ的表达水平:自术后第3天起经鞘内多次给药,与生理盐水组相比,应用小胶质细胞抑制剂—米诺环素后大鼠PWT、脊髓T细胞数量和IFN-γ的表达水平均无显著变化;运用星形胶质细胞抑制剂—氟代柠檬酸后,PWT明显升高,IFN-γ的表达水平显著下降,而T细胞的浸润数目无明显变化。三、炎性痛大鼠脊髓IFN-γ表达与星形胶质细胞活化的相关性IFN-γ调节疼痛及星形胶质细胞活化:与磷酸盐缓冲液组相比,鞘内连续给予IFN-γ中和抗体可明显提高PWT,并下调星形胶质细胞GFAP的表达。而naive大鼠经鞘内给予重组IFN-γ后,与生理盐水组相比,能够引起PWT持续降低,并上调星形胶质细胞GFAP表达。体外IFN-γ孵育原代星形胶质细胞可增加星形胶质细胞中NF-κBp65的磷酸化水平。结论外周炎症发生后,浸润至脊髓的T细胞数量与星形胶质细胞的活化表现出相似的动态变化。参与疼痛调节的T细胞亚型最可能为Th1,改变脊髓IFN-γ的表达水平可直接引起痛行为学变化,结合体内外实验证实IFN-γ对星形胶质细胞具有直接激活作用。星形胶质细胞参与了炎性疼痛的维持,并调节脊髓IFN-γ的表达水平。总而言之,本研究证实Th1细胞可能通过分泌IFN-γ激活星形胶质细胞参与炎性痛的发展,反之,星形胶质细胞活化后可调控Th1细胞产生IFN-γ,协同推动炎性痛慢性化的形成。
[Abstract]:Objective Pathological pain (also known as chronic pain) has become the world's largest burden of disease, inflammatory pain is one of the main types of clinical pathological pain. Previous studies have found that after tissue injury or inflammation, spinal microglia and astrocytes synthesize, release a variety of inflammatory cytokines, chemokines and neurotrophic factors. Factor and so on, mutual influence, constitutes the local inflammation microenvironment, finally strengthens the excitatory synaptic transmission, namely the central sensitization formation, the organism appears the hyperalgesia and the hyperalgesia abnormality, but the central sensitization long-term maintenance or the pain chronicity key mechanism is still unclear. Th1 cells secreting IFN-gamma and Th17 cells secreting IL-17 are involved in the pathophysiological process of multiple sclerosis. The effect of T cells on neuropathic pain has been gradually discovered and is now thought to be associated with the chronicity of neuropathic pain. The specific molecular mechanism of T cells regulating pain and their effects on the occurrence and development of inflammatory pain are still poorly understood. Peripheral blood T cells involved in the regulation of pathological pain in the spinal cord must be recruited from the central spinal cord and need to pass through the blood-central nervous system. Many inflammatory factors such as TNF-a, IL-1beta and CCL2 produced by the activation of spinal cord glial cells may increase the permeability of the blood-central nervous system barrier and enhance the chemotaxis of peripheral immune cells after inflammatory injury. Astrocytes are involved in the formation of the blood-central nervous system barrier. It has been found that microglia and astrocytes can up-regulate the expression of MHC class II molecules under suitable stimulation such as inflammation, which may be antigen presenting cells in the central nervous system. Therefore, we speculate that the activation of glia, especially astrocytes, may play an immune role in the infiltration of T cells into the spinal cord and maintain pathological changes. In order to verify the above hypothesis, we used the inflammatory pain model of uniarthritis rats and primary astrocytes, using immunofluorescence histochemistry, flow cytometry, protein immunoblotting and other experimental methods to explore the contribution of T cells to inflammatory pain and its molecular mechanism. 1. To observe the number of T cells and the activation of glial cells in the spinal cord lumbar enlargement of rats with monoarthritis (MA), establish the inflammatory pain model of rats with monoarthritis (MA). At the same time, the expression levels of ionic calcium-binding receptor molecule-1 (Iba1) and glial fibrillary acidic protein (GFAP) in microglia were detected to observe and compare the dynamic changes of T cell number and glial cell activation. To investigate the relationship between the activation status of spinal cord glial cells and the number and activity of spinal cord T cells, a MA model was established. The activation of microglia and astrocytes was inhibited by pharmacological means. The number of T cells and the expression of IFN-gamma in PWT, spinal cord lumbar enlargement were detected. To investigate the correlation between IFN-gamma expression and astrocyte activation in spinal cord of rats with inflammatory pain. The levels of IFN-gamma expression in spinal cord of MA and naive rats were changed by pharmacological methods. The Paw withdrawal threshold (PWT) and GFAP expression in astrocyte were detected after incubation with IFN-gamma. The phosphorylation level of NF-kappa Bp65 in primary astrocytes was determined to clarify the regulatory effect of IFN-gamma on astrocytes. Results 1. T cell infiltration and glial activation in spinal cord after inflammation induced by the number of T cells and the activation of glial cells in spinal cord lumbar enlargement in uniarthritic rats: The number of T cells in spinal cord lumbar enlargement was 7 days and 10 days after inflammation. IFN-gamma was mainly co-labeled with T-cell-labeled CD3, while Th17-labeled IL-17 was mainly co-labeled with astrocyte-labeled GFAP. On the 7th and 10th day after operation, the expression of microglia Iba1 and astrocyte GFAP was up-regulated, and the expression of IFN-gamma was up-regulated in both groups. Iba1 and GFAP were up-regulated more significantly on the 7th and 10th day, respectively. 2. The relationship between the activation of spinal cord glial cells and the number and activity of spinal cord T cells astrocytes regulated the expression of spinal cord T cell-related factor IFN-gamma. Since the 3rd day after operation, microglial inhibitor Mi was used more frequently in the intrathecal administration than in the saline group. There were no significant changes in PWT, the number of T cells and the expression of IFN-gamma in the spinal cord of rats after norcycline treatment. After the use of astrocyte inhibitor fluorocitrate, the expression of PWT increased significantly, the expression of IFN-gamma decreased significantly, but the number of T cells infiltrated did not change significantly. 3. The expression of IFN-gamma and the activation of astrocytes in the spinal cord of rats with inflammatory pain. Relevant IFN-gamma regulates pain and astrocyte activation: Compared with phosphate buffer group, continuous intrathecal administration of IFN-gamma neutralizing antibody can significantly increase PWT and down-regulate GFAP expression in astrocytes. However, after intrathecal administration of recombinant IFN-gamma in naive rats, PWT is continuously decreased and astrogel is up-regulated. In vitro IFN-gamma incubation of primary astrocytes can increase the phosphorylation level of NF-kappa Bp65 in astrocytes.Conclusion After peripheral inflammation, the number of T cells infiltrating into the spinal cord and the activation of astrocytes show similar dynamic changes.T cell subtypes involved in pain regulation are most likely to be Th1, which alters the spinal cord. The expression level of IFN-gamma in the spinal cord can directly induce pain behavior changes. Combined with in vivo and in vitro experiments, IFN-gamma has a direct activation effect on astrocytes. Astrocytes participate in the maintenance of inflammatory pain and regulate the expression level of IFN-gamma in the spinal cord. Plasmacytes participate in the development of inflammatory pain. On the contrary, the activation of astrocytes can regulate the production of IFN-gamma by Th1 cells and promote the formation of chronic inflammatory pain.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614
本文编号：2226046
[Abstract]:Objective Pathological pain (also known as chronic pain) has become the world's largest burden of disease, inflammatory pain is one of the main types of clinical pathological pain. Previous studies have found that after tissue injury or inflammation, spinal microglia and astrocytes synthesize, release a variety of inflammatory cytokines, chemokines and neurotrophic factors. Factor and so on, mutual influence, constitutes the local inflammation microenvironment, finally strengthens the excitatory synaptic transmission, namely the central sensitization formation, the organism appears the hyperalgesia and the hyperalgesia abnormality, but the central sensitization long-term maintenance or the pain chronicity key mechanism is still unclear. Th1 cells secreting IFN-gamma and Th17 cells secreting IL-17 are involved in the pathophysiological process of multiple sclerosis. The effect of T cells on neuropathic pain has been gradually discovered and is now thought to be associated with the chronicity of neuropathic pain. The specific molecular mechanism of T cells regulating pain and their effects on the occurrence and development of inflammatory pain are still poorly understood. Peripheral blood T cells involved in the regulation of pathological pain in the spinal cord must be recruited from the central spinal cord and need to pass through the blood-central nervous system. Many inflammatory factors such as TNF-a, IL-1beta and CCL2 produced by the activation of spinal cord glial cells may increase the permeability of the blood-central nervous system barrier and enhance the chemotaxis of peripheral immune cells after inflammatory injury. Astrocytes are involved in the formation of the blood-central nervous system barrier. It has been found that microglia and astrocytes can up-regulate the expression of MHC class II molecules under suitable stimulation such as inflammation, which may be antigen presenting cells in the central nervous system. Therefore, we speculate that the activation of glia, especially astrocytes, may play an immune role in the infiltration of T cells into the spinal cord and maintain pathological changes. In order to verify the above hypothesis, we used the inflammatory pain model of uniarthritis rats and primary astrocytes, using immunofluorescence histochemistry, flow cytometry, protein immunoblotting and other experimental methods to explore the contribution of T cells to inflammatory pain and its molecular mechanism. 1. To observe the number of T cells and the activation of glial cells in the spinal cord lumbar enlargement of rats with monoarthritis (MA), establish the inflammatory pain model of rats with monoarthritis (MA). At the same time, the expression levels of ionic calcium-binding receptor molecule-1 (Iba1) and glial fibrillary acidic protein (GFAP) in microglia were detected to observe and compare the dynamic changes of T cell number and glial cell activation. To investigate the relationship between the activation status of spinal cord glial cells and the number and activity of spinal cord T cells, a MA model was established. The activation of microglia and astrocytes was inhibited by pharmacological means. The number of T cells and the expression of IFN-gamma in PWT, spinal cord lumbar enlargement were detected. To investigate the correlation between IFN-gamma expression and astrocyte activation in spinal cord of rats with inflammatory pain. The levels of IFN-gamma expression in spinal cord of MA and naive rats were changed by pharmacological methods. The Paw withdrawal threshold (PWT) and GFAP expression in astrocyte were detected after incubation with IFN-gamma. The phosphorylation level of NF-kappa Bp65 in primary astrocytes was determined to clarify the regulatory effect of IFN-gamma on astrocytes. Results 1. T cell infiltration and glial activation in spinal cord after inflammation induced by the number of T cells and the activation of glial cells in spinal cord lumbar enlargement in uniarthritic rats: The number of T cells in spinal cord lumbar enlargement was 7 days and 10 days after inflammation. IFN-gamma was mainly co-labeled with T-cell-labeled CD3, while Th17-labeled IL-17 was mainly co-labeled with astrocyte-labeled GFAP. On the 7th and 10th day after operation, the expression of microglia Iba1 and astrocyte GFAP was up-regulated, and the expression of IFN-gamma was up-regulated in both groups. Iba1 and GFAP were up-regulated more significantly on the 7th and 10th day, respectively. 2. The relationship between the activation of spinal cord glial cells and the number and activity of spinal cord T cells astrocytes regulated the expression of spinal cord T cell-related factor IFN-gamma. Since the 3rd day after operation, microglial inhibitor Mi was used more frequently in the intrathecal administration than in the saline group. There were no significant changes in PWT, the number of T cells and the expression of IFN-gamma in the spinal cord of rats after norcycline treatment. After the use of astrocyte inhibitor fluorocitrate, the expression of PWT increased significantly, the expression of IFN-gamma decreased significantly, but the number of T cells infiltrated did not change significantly. 3. The expression of IFN-gamma and the activation of astrocytes in the spinal cord of rats with inflammatory pain. Relevant IFN-gamma regulates pain and astrocyte activation: Compared with phosphate buffer group, continuous intrathecal administration of IFN-gamma neutralizing antibody can significantly increase PWT and down-regulate GFAP expression in astrocytes. However, after intrathecal administration of recombinant IFN-gamma in naive rats, PWT is continuously decreased and astrogel is up-regulated. In vitro IFN-gamma incubation of primary astrocytes can increase the phosphorylation level of NF-kappa Bp65 in astrocytes.Conclusion After peripheral inflammation, the number of T cells infiltrating into the spinal cord and the activation of astrocytes show similar dynamic changes.T cell subtypes involved in pain regulation are most likely to be Th1, which alters the spinal cord. The expression level of IFN-gamma in the spinal cord can directly induce pain behavior changes. Combined with in vivo and in vitro experiments, IFN-gamma has a direct activation effect on astrocytes. Astrocytes participate in the maintenance of inflammatory pain and regulate the expression level of IFN-gamma in the spinal cord. Plasmacytes participate in the development of inflammatory pain. On the contrary, the activation of astrocytes can regulate the production of IFN-gamma by Th1 cells and promote the formation of chronic inflammatory pain.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614
【共引文献】
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