【摘要】：近年來,有關(guān)炎性反應(yīng)受到神經(jīng)回路調(diào)控的發(fā)現(xiàn)將免疫學(xué)和神經(jīng)生物學(xué)聯(lián)系到了一起。這一發(fā)現(xiàn)的意義在于免疫系統(tǒng)將不再被認(rèn)作是一個完全自主的獨(dú)立系統(tǒng),而這種炎癥反應(yīng)的神經(jīng)系統(tǒng)調(diào)控通路被稱為膽堿抗炎癥通路。a7 nAChR,是乙酰膽堿受體家族的一個成員,它在神經(jīng)系統(tǒng)中廣泛分布。該家族成員在神經(jīng)元細(xì)胞上形成同五聚體或者異五聚體受體,并作為配體控制的離子通道介導(dǎo)突觸中信號的快速傳遞。除了神經(jīng)系統(tǒng),a7 nAChR在免疫細(xì)胞中廣泛表達(dá),現(xiàn)已在單核、巨噬、T、B淋巴細(xì)胞和樹突狀細(xì)胞中證實(shí)了其存在。使用a7 nAChR的非選擇性激活劑(如nicotine)或者選擇性激活劑(如choline或GTS-21)能夠在單核和巨噬細(xì)胞中活化多個細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)級聯(lián)反應(yīng),下調(diào)核轉(zhuǎn)錄因子NF-κB的活性以及TLR4的表達(dá),并且抑制炎性因子的轉(zhuǎn)錄。 本實(shí)驗(yàn)應(yīng)用膠原誘導(dǎo)關(guān)節(jié)炎(collagen induced arthritis, CIA)模型小鼠作為實(shí)驗(yàn)?zāi)Ｐ?檢測了煙堿干預(yù)對CIA小鼠的免疫調(diào)節(jié)作用。結(jié)果顯示,煙堿干預(yù)能夠有效地減緩模型小鼠關(guān)節(jié)炎病程,減輕炎癥細(xì)胞浸潤、關(guān)節(jié)軟骨侵蝕等關(guān)節(jié)炎癥狀；而且流式結(jié)果顯示煙堿干預(yù)能夠降低模型小鼠脾臟中CD4+IL-17A+細(xì)胞比例(12.53% vs.7.63%);減弱炎癥相關(guān)細(xì)胞因子IL-17A、IL-21、IL-6 mRNA的表達(dá)。并且煙堿干預(yù)能夠降低模型小鼠血漿中炎癥細(xì)胞因子TNF-a含量。 同時,為了進(jìn)一步了解乙酰膽堿受體在CIA模型小鼠免疫系統(tǒng)中的作用機(jī)制,本研究檢測了體外培養(yǎng)Th17細(xì)胞的乙酰膽堿受體(a7 nAChR)表達(dá),結(jié)果顯示在所有檢測細(xì)胞中,乙酰膽堿受體(a7 nAChR)陽性Th17細(xì)胞為8.49%,占IL-17A+細(xì)胞總數(shù)的25.64%。隨后,我們采用了抗體四聚體聯(lián)合磁珠分選方法,對Th17條件培養(yǎng)細(xì)胞進(jìn)行了分選純化,純化后IL-17A+細(xì)胞達(dá)到58.49%。采用煙堿對純化后Th17培養(yǎng)細(xì)胞進(jìn)行處理,流式檢測結(jié)果發(fā)現(xiàn)相比對照組,加入10-6M煙堿的培養(yǎng)細(xì)胞其細(xì)胞內(nèi)IL-17A熒光強(qiáng)度明顯下降。進(jìn)一步的流式CBA檢測表明煙堿處理培養(yǎng)細(xì)胞的培養(yǎng)上清中IL-17A的含量低于對照組(546.99±17.73 pg/ml vs.669.36±21.15 pg/ml)。.. 本研究將乙酰膽堿受體與膠原誘導(dǎo)關(guān)節(jié)炎模型相關(guān)聯(lián),為乙酰膽堿受體為靶向的治療方式提供實(shí)驗(yàn)依據(jù)。同時,初步探討了乙酰膽堿受體活化對Th17細(xì)胞功能的影響,為乙酰膽堿受體調(diào)控炎癥機(jī)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 鹵17.73 pg/ml vs.669.36 鹵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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

【共引文獻(xiàn)】

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本文編號：2455107