IFN-γ介導(dǎo)T細(xì)胞參與炎性痛慢性化機(jī)制的研究
發(fā)布時(shí)間：2018-09-06 10:17
【摘要】：研究目的病理性疼痛(又稱為慢性痛)已經(jīng)成為全球最大疾病負(fù)擔(dān),炎性痛是臨床病理性疼痛的主要類型之一。既往研究發(fā)現(xiàn),在組織損傷或炎癥發(fā)生后,脊髓小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞合成、釋放多種促炎細(xì)胞因子、趨化因子和神經(jīng)營養(yǎng)因子等,彼此相互影響,構(gòu)成局部炎癥微環(huán)境,最終增強(qiáng)興奮性突觸傳遞,即中樞敏化的形成,機(jī)體出現(xiàn)痛覺過敏和痛覺異常,但中樞敏化的長(zhǎng)期維持或疼痛慢性化的關(guān)鍵機(jī)制仍不清楚。神經(jīng)系統(tǒng)退行性疾病多由自身抗原特異性地T細(xì)胞介導(dǎo),且常常伴有疼痛癥狀,如分泌IFN-γ的Th1細(xì)胞和分泌IL-17的Th17細(xì)胞均參與多發(fā)性硬化的病理過程。T細(xì)胞對(duì)神經(jīng)病理性疼痛的影響逐漸被發(fā)現(xiàn),目前認(rèn)為其作用與神經(jīng)病理性疼痛的慢性化相關(guān),至于具體的T細(xì)胞亞型,現(xiàn)有證據(jù)趨向于Th1細(xì)胞,Th17細(xì)胞還有待進(jìn)一步研究。T細(xì)胞調(diào)控疼痛的具體分子作用機(jī)制,以及它們對(duì)炎性痛的發(fā)生、發(fā)展是否有影響及作用機(jī)制目前研究較少。外周血T細(xì)胞參與脊髓對(duì)病理性疼痛的調(diào)控,就必須向脊髓中樞募集,需要通過血-中樞神經(jīng)系統(tǒng)屏障進(jìn)入脊髓。炎性損傷發(fā)生后,脊髓膠質(zhì)細(xì)胞激活產(chǎn)生的多種炎癥因子如TNF-α、IL-1β、CCL2等可能會(huì)增加血-中樞神經(jīng)系統(tǒng)屏障的通透性,提高對(duì)外周免疫細(xì)胞的趨化作用,其中星形膠質(zhì)細(xì)胞更是參與了血-中樞神經(jīng)系統(tǒng)屏障的構(gòu)成。此外有研究發(fā)現(xiàn)小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞在炎癥等適宜刺激下可上調(diào)表達(dá)MHC-Ⅱ類分子,可能是中樞神經(jīng)系統(tǒng)的抗原遞呈細(xì)胞。據(jù)此我們推測(cè)膠質(zhì)細(xì)胞的活化尤其是星形膠質(zhì)細(xì)胞的活化可能為T細(xì)胞浸潤至脊髓發(fā)揮免疫效應(yīng)、維持病理性疼痛提供了必要條件。研究方法為驗(yàn)證以上推測(cè),本課題利用單關(guān)節(jié)炎大鼠炎性痛模型和原代星形膠質(zhì)細(xì)胞,運(yùn)用免疫熒光組織化學(xué)方法、流式細(xì)胞術(shù)、蛋白質(zhì)免疫印跡等實(shí)驗(yàn)方法,依以下步驟初步探討T細(xì)胞對(duì)炎性痛的貢獻(xiàn)、分子機(jī)制,以及膠質(zhì)細(xì)胞對(duì)它的調(diào)節(jié)作用。一、觀察單關(guān)節(jié)炎大鼠脊髓腰膨大中T細(xì)胞數(shù)量及膠質(zhì)細(xì)胞活化情況建立單關(guān)節(jié)炎大鼠(monoarthritis rats,MA)炎性痛模型,分離MA組和假手術(shù)組大鼠致炎后第7天、10天的脊髓腰膨大,檢測(cè)T細(xì)胞數(shù)量、分布位置及活化情況,同時(shí)檢測(cè)小膠質(zhì)細(xì)胞離子型鈣結(jié)合受體分子-1(Ionized calcium-binding adaptor molecule-1,Iba1)和星形膠質(zhì)細(xì)胞膠質(zhì)纖維酸性蛋白(Glial fibrillary acidic protein,GFAP)的表達(dá)水平,觀察與比較T細(xì)胞數(shù)量與膠質(zhì)細(xì)胞活化的動(dòng)態(tài)變化。二、分析探討脊髓膠質(zhì)細(xì)胞的活化狀態(tài)與脊髓T細(xì)胞數(shù)量及活性的關(guān)系建立MA模型,通過藥理學(xué)手段抑制小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞的活化,檢測(cè)PWT,以及脊髓腰膨大中T細(xì)胞數(shù)量及IFN-γ的表達(dá)水平,初步探討膠質(zhì)細(xì)胞活化對(duì)T細(xì)胞的調(diào)節(jié)作用。三、分析探討炎性痛大鼠脊髓IFN-γ表達(dá)與星形膠質(zhì)細(xì)胞活化的相關(guān)性通過藥理學(xué)手段改變MA及naive組大鼠脊髓IFN-γ的表達(dá)水平后檢測(cè)機(jī)械縮腿反應(yīng)閾值(Paw withdrawal threshold,PWT),以及星形膠質(zhì)細(xì)胞GFAP的表達(dá)水平。并進(jìn)一步檢測(cè)經(jīng)IFN-γ孵育后的原代星形膠質(zhì)細(xì)胞中NF-κBp65的磷酸化水平,明確IFN-γ對(duì)星形膠質(zhì)細(xì)胞的調(diào)節(jié)作用。結(jié)果一、單關(guān)節(jié)炎大鼠脊髓腰膨大中T細(xì)胞數(shù)量及膠質(zhì)細(xì)胞活化情況致炎后脊髓T細(xì)胞浸潤及膠質(zhì)細(xì)胞活化:脊髓腰膨大中的T細(xì)胞數(shù)目在致炎后第7天、10天明顯增加,并且在第10天增加更顯著。T細(xì)胞主要分布在脊髓背角。IFN-γ主要與T細(xì)胞標(biāo)記CD3共標(biāo),而Th17標(biāo)志性細(xì)胞因子IL-17主要與星形膠質(zhì)細(xì)胞標(biāo)記GFAP共標(biāo)。在術(shù)后第7天和第10天,小膠質(zhì)細(xì)胞Iba1和星形膠質(zhì)細(xì)胞GFAP表達(dá)水平均上調(diào),其中Iba1和GFAP分別在第7天、10天上調(diào)更顯著。二、脊髓膠質(zhì)細(xì)胞的活化狀態(tài)與脊髓T細(xì)胞數(shù)量及活性的關(guān)系星形膠質(zhì)細(xì)胞調(diào)節(jié)脊髓T細(xì)胞相關(guān)因子IFN-γ的表達(dá)水平:自術(shù)后第3天起經(jīng)鞘內(nèi)多次給藥,與生理鹽水組相比,應(yīng)用小膠質(zhì)細(xì)胞抑制劑—米諾環(huán)素后大鼠PWT、脊髓T細(xì)胞數(shù)量和IFN-γ的表達(dá)水平均無顯著變化;運(yùn)用星形膠質(zhì)細(xì)胞抑制劑—氟代檸檬酸后,PWT明顯升高,IFN-γ的表達(dá)水平顯著下降,而T細(xì)胞的浸潤數(shù)目無明顯變化。三、炎性痛大鼠脊髓IFN-γ表達(dá)與星形膠質(zhì)細(xì)胞活化的相關(guān)性IFN-γ調(diào)節(jié)疼痛及星形膠質(zhì)細(xì)胞活化:與磷酸鹽緩沖液組相比,鞘內(nèi)連續(xù)給予IFN-γ中和抗體可明顯提高PWT,并下調(diào)星形膠質(zhì)細(xì)胞GFAP的表達(dá)。而naive大鼠經(jīng)鞘內(nèi)給予重組IFN-γ后,與生理鹽水組相比,能夠引起PWT持續(xù)降低,并上調(diào)星形膠質(zhì)細(xì)胞GFAP表達(dá)。體外IFN-γ孵育原代星形膠質(zhì)細(xì)胞可增加星形膠質(zhì)細(xì)胞中NF-κBp65的磷酸化水平。結(jié)論外周炎癥發(fā)生后,浸潤至脊髓的T細(xì)胞數(shù)量與星形膠質(zhì)細(xì)胞的活化表現(xiàn)出相似的動(dòng)態(tài)變化。參與疼痛調(diào)節(jié)的T細(xì)胞亞型最可能為Th1,改變脊髓IFN-γ的表達(dá)水平可直接引起痛行為學(xué)變化,結(jié)合體內(nèi)外實(shí)驗(yàn)證實(shí)IFN-γ對(duì)星形膠質(zhì)細(xì)胞具有直接激活作用。星形膠質(zhì)細(xì)胞參與了炎性疼痛的維持,并調(diào)節(jié)脊髓IFN-γ的表達(dá)水平。總而言之,本研究證實(shí)Th1細(xì)胞可能通過分泌IFN-γ激活星形膠質(zhì)細(xì)胞參與炎性痛的發(fā)展,反之,星形膠質(zhì)細(xì)胞活化后可調(diào)控Th1細(xì)胞產(chǎn)生IFN-γ,協(xié)同推動(dòng)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R614
本文編號(hào)：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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R614
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