烟碱对胶原诱导关节炎小鼠调节作用及烟碱对体外诱导Th17细胞分化和功能影响的初步研究
发布时间：2019-04-09 10:39
【摘要】：近年来,有关炎性反应受到神经回路调控的发现将免疫学和神经生物学联系到了一起。这一发现的意义在于免疫系统将不再被认作是一个完全自主的独立系统,而这种炎症反应的神经系统调控通路被称为胆碱抗炎症通路。a7 nAChR,是乙酰胆碱受体家族的一个成员,它在神经系统中广泛分布。该家族成员在神经元细胞上形成同五聚体或者异五聚体受体,并作为配体控制的离子通道介导突触中信号的快速传递。除了神经系统,a7 nAChR在免疫细胞中广泛表达,现已在单核、巨噬、T、B淋巴细胞和树突状细胞中证实了其存在。使用a7 nAChR的非选择性激活剂(如nicotine)或者选择性激活剂(如choline或GTS-21)能够在单核和巨噬细胞中活化多个细胞内信号转导级联反应,下调核转录因子NF-κB的活性以及TLR4的表达,并且抑制炎性因子的转录。 本实验应用胶原诱导关节炎(collagen induced arthritis, CIA)模型小鼠作为实验模型,检测了烟碱干预对CIA小鼠的免疫调节作用。结果显示,烟碱干预能够有效地减缓模型小鼠关节炎病程,减轻炎症细胞浸润、关节软骨侵蚀等关节炎症状；而且流式结果显示烟碱干预能够降低模型小鼠脾脏中CD4+IL-17A+细胞比例(12.53% vs.7.63%);减弱炎症相关细胞因子IL-17A、IL-21、IL-6 mRNA的表达。并且烟碱干预能够降低模型小鼠血浆中炎症细胞因子TNF-a含量。 同时,为了进一步了解乙酰胆碱受体在CIA模型小鼠免疫系统中的作用机制,本研究检测了体外培养Th17细胞的乙酰胆碱受体(a7 nAChR)表达,结果显示在所有检测细胞中,乙酰胆碱受体(a7 nAChR)阳性Th17细胞为8.49%,占IL-17A+细胞总数的25.64%。随后,我们采用了抗体四聚体联合磁珠分选方法,对Th17条件培养细胞进行了分选纯化,纯化后IL-17A+细胞达到58.49%。采用烟碱对纯化后Th17培养细胞进行处理,流式检测结果发现相比对照组,加入10-6M烟碱的培养细胞其细胞内IL-17A荧光强度明显下降。进一步的流式CBA检测表明烟碱处理培养细胞的培养上清中IL-17A的含量低于对照组(546.99±17.73 pg/ml vs.669.36±21.15 pg/ml)。.. 本研究将乙酰胆碱受体与胶原诱导关节炎模型相关联,为乙酰胆碱受体为靶向的治疗方式提供实验依据。同时,初步探讨了乙酰胆碱受体活化对Th17细胞功能的影响,为乙酰胆碱受体调控炎症机制提供新的研究思路。
[Abstract]:In recent years, immunology and neurobiology have been linked to the discovery that inflammatory responses are regulated by neural circuits. The significance of this discovery is that the immune system will no longer be recognized as a completely autonomous, independent system, and that the neural regulatory pathway of the inflammatory response is known as the choline anti-inflammatory pathway. A 7 nAChR, is a member of the acetylcholine receptor family. It is widely distributed in the nervous system. Members of the family form homopolymeric or heteropentameric receptors on neurons and act as ligand-controlled ion channels that mediate the rapid transmission of signals in synapses. In addition to the nervous system, a7 nAChR is widely expressed in immune cells and has been confirmed to exist in monocytes, macrophages, T, B lymphocytes and dendritic cells. Non-selective activators (such as nicotine) or selective activators (such as choline or GTS-21) of a7 nAChR can activate multiple intracellular signal transduction cascades in monocytes and macrophages. Down-regulate the activity of nuclear transcription factor NF- kappa B and the expression of TLR4, and inhibit the transcription of inflammatory factors. The immunomodulatory effect of nicotine intervention on (collagen induced arthritis, CIA) mice with collagen-induced arthritis was studied in this study. The results showed that nicotine intervention could effectively slow down the course of arthritis, reduce inflammatory cell infiltration, articular cartilage erosion and other arthritis symptoms. The results of flow cytometry showed that nicotine could decrease the percentage of CD4 IL-17A cells (12.53% vs.7.63%) and decrease the expression of IL-17A,IL-21,IL-6 mRNA in spleen of the model mice. Nicotine intervention could decrease the content of inflammatory cytokine TNF-a in plasma of model mice. At the same time, in order to further understand the mechanism of acetylcholine receptor in the immune system of CIA model mice, the expression of acetylcholine receptor (a7 nAChR) in Th17 cells cultured in vitro was detected, and the results showed that the expression of acetylcholine receptor (a7 nAChR) was detected in all the detected cells. Acetylcholine receptor (a 7 nAChR) positive Th17 cells were 8.49%, accounting for 25.64% of the total IL-17A cells. Then, we used antibody tetramer combined with magnetic beads to separate and purify the cells cultured in Th17 condition. After purification, the IL-17A cells reached 58.49%. The cultured cells of purified Th17 were treated with nicotine. The results of flow cytometry showed that the intracellular IL-17A fluorescence intensity of cultured cells with 10 m nicotine decreased significantly compared with the control group. Further flow CBA analysis showed that the content of IL-17A in the supernatant of cultured cells treated with nicotine was lower than that of the control group (546.99 卤17.73 pg/ml vs.669.36 卤21.15 pg/ml). In this study, acetylcholine receptor was associated with collagen-induced arthritis model, which provided experimental basis for acetylcholine receptor as targeted therapy. At the same time, the effect of acetylcholine receptor activation on the function of Th17 cells was discussed, which provided a new way to study the mechanism of acetylcholine receptor regulating inflammation.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
[Abstract]:In recent years, immunology and neurobiology have been linked to the discovery that inflammatory responses are regulated by neural circuits. The significance of this discovery is that the immune system will no longer be recognized as a completely autonomous, independent system, and that the neural regulatory pathway of the inflammatory response is known as the choline anti-inflammatory pathway. A 7 nAChR, is a member of the acetylcholine receptor family. It is widely distributed in the nervous system. Members of the family form homopolymeric or heteropentameric receptors on neurons and act as ligand-controlled ion channels that mediate the rapid transmission of signals in synapses. In addition to the nervous system, a7 nAChR is widely expressed in immune cells and has been confirmed to exist in monocytes, macrophages, T, B lymphocytes and dendritic cells. Non-selective activators (such as nicotine) or selective activators (such as choline or GTS-21) of a7 nAChR can activate multiple intracellular signal transduction cascades in monocytes and macrophages. Down-regulate the activity of nuclear transcription factor NF- kappa B and the expression of TLR4, and inhibit the transcription of inflammatory factors. The immunomodulatory effect of nicotine intervention on (collagen induced arthritis, CIA) mice with collagen-induced arthritis was studied in this study. The results showed that nicotine intervention could effectively slow down the course of arthritis, reduce inflammatory cell infiltration, articular cartilage erosion and other arthritis symptoms. The results of flow cytometry showed that nicotine could decrease the percentage of CD4 IL-17A cells (12.53% vs.7.63%) and decrease the expression of IL-17A,IL-21,IL-6 mRNA in spleen of the model mice. Nicotine intervention could decrease the content of inflammatory cytokine TNF-a in plasma of model mice. At the same time, in order to further understand the mechanism of acetylcholine receptor in the immune system of CIA model mice, the expression of acetylcholine receptor (a7 nAChR) in Th17 cells cultured in vitro was detected, and the results showed that the expression of acetylcholine receptor (a7 nAChR) was detected in all the detected cells. Acetylcholine receptor (a 7 nAChR) positive Th17 cells were 8.49%, accounting for 25.64% of the total IL-17A cells. Then, we used antibody tetramer combined with magnetic beads to separate and purify the cells cultured in Th17 condition. After purification, the IL-17A cells reached 58.49%. The cultured cells of purified Th17 were treated with nicotine. The results of flow cytometry showed that the intracellular IL-17A fluorescence intensity of cultured cells with 10 m nicotine decreased significantly compared with the control group. Further flow CBA analysis showed that the content of IL-17A in the supernatant of cultured cells treated with nicotine was lower than that of the control group (546.99 卤17.73 pg/ml vs.669.36 卤21.15 pg/ml). In this study, acetylcholine receptor was associated with collagen-induced arthritis model, which provided experimental basis for acetylcholine receptor as targeted therapy. At the same time, the effect of acetylcholine receptor activation on the function of Th17 cells was discussed, which provided a new way to study the mechanism of acetylcholine receptor regulating inflammation.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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