乙酰胆碱M受体调控小胶质细胞免疫应答作用研究

发布时间：2018-06-18 00:56

  本文选题：小胶质细胞 + 免疫应答　； 参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：阿尔兹海默症(Alzheimer’s disease,AD)是发生在老年期及老年前期的一种慢性退化性脑变疾病,以进行性记忆减退、认知障碍、人格改变为临床表现。在人口老龄化的当代社会,AD成为继心脑血管疾病,肿瘤和脑卒中的第四大杀手,威胁着老年人身体健康,降低了生活质量,加重家庭和社会负担。AD发病原因复杂多样,目前具体发病机制仍不明确。近年研究发现,神经炎症反应是AD的一个重要的致病原因。AD患者脑内小胶质细胞(Microglia,MG)高度活化,炎性因子含量增高。作为神经炎性的始作俑者,MG免疫功能的调控成为AD治疗的新靶点,其免疫功能的研究也跻身于神经退行性疾病领域的研究热点。MG在CNS中的作用是把双刃剑,激活的MG吞噬细胞残渣,通过抗原递呈功能、细胞因子分泌参与免疫应答反应,移除病原体。然而,MG过度激活导致CNS发生慢性炎症,加重大脑损伤。因此,严格调控MG免疫反应是防止神经炎性发生的关键。乙酰胆碱是一类参与中枢大脑学习、认知、记忆等的重要神经递质,乙酰胆碱作用的受体包括毒蕈碱型胆碱受体(Muscarinic acetylcholine receptors,mAChRs)和烟碱型胆碱受体(Nicotinic acetylcholine receptor,nAChRs)。近年来研究表明,乙酰胆碱系统中nAChRs在MG介导的神经炎性发挥作用,а7烟碱型受体(а7nAChR)在MG表达,激活а7nAChR抑制MG的炎性反应。然而,mAChRs在MG的表达情况以及是否参与MG的炎性调控尚不清楚。本实验室前期研究发现,SD大鼠长期腹腔注射m AChRs非选择性拮抗剂盐酸苯海索(Trihexyphenidyl hydrochloride,THP)促进大脑海马和皮层的MG增生和激活,上调MG的CD68表达同时伴随M1受体表达增加,并且两者存在共定位,暗示M受体介导THP对MG活化的调控作用。在本研究中,我们将首次确证mAChRs在MG的表达情况,并且观察mAChRs对MG免疫应答功能的调控作用。本研究的开展利于探讨m AChRs与神经炎性的关系,开拓了其影响AD发展的新机制研究,有利于寻找治疗AD的新靶点。我们首先建立原代大鼠皮层小胶质细胞培养方法,在体外条件下,确证mAChRs在小胶质细胞表达情况;利用mAChRs非选择性激动剂氧化震颤素(Oxotremorine,OX)、mAChRs非选择拮抗剂阿托品和M1受体阻断剂毒蕈碱毒素7(Muscarinic Toxin 7,MT7)三种工具药,研究mAChRs对小胶质细胞抗原递呈功能、小胶质细胞活化和免疫炎症介质释放的调控作用。方法:(1)建立大鼠皮层原代小胶质细胞培养方法:将出生12 h内的SD大鼠的皮层神经胶质细胞混合培养8-9天,小胶质细胞分层明显且数量达到最多,利用胰酶温和消化法和震摇法进行小胶质细胞分离纯化;纯化的小胶质细胞培养3天后基本恢复静息状态,通过小胶质细胞标记分子Iba1免疫荧光染色进行鉴定。(2)mAChRs在小胶质细胞表达研究:利用RT-PCR、Western-blot技术从mRNA和蛋白水平检测mAChRs在小胶质细胞表达情况。(3)mAChRs对小胶质细胞抗原递呈功能影响研究:mAChRs非选择性激动剂OX处理小胶质细胞,利用激光共聚焦扫描显微镜(Confocal laser scanning microscope,CLSM)结合免疫荧光、流式细胞术观察OX对主要组织相容性抗原分子Ι(Major Histocompatibility ComplexΙ,MHCΙ)表达影响;荧光定量PCR检测OX对小胶质细胞抗原递呈分子相关基因Rt1-Aw2、β2m和Tap1 mRNA表达影响。(4)M1受体参与mAChRs调控小胶质细胞抗原递呈作用研究:利用M1受体阻断剂MT7,应用免疫荧光结合CLSM、Western-blot和荧光定量PCR检测MT7对小胶质细胞MHCΙ分子和M1受体表达的变化。(5)mAChRs对小胶质细胞活化的影响:免疫荧光结合CLSM和Western-blot检测OX单独给药以及MT7和OX伴随给药对小胶质细胞激活分子CD68和CD11b表达影响。(6)mAChRs对小胶质细胞炎症介质释放影响:Luminex技术检测OX对小胶质细胞IL-1β、TNF-а和IL-10释放的影响。结果:(1)改进了原代小胶质细胞的培养方法,小胶质细胞Iba1阳性率高达95%;观察了小胶质细胞生长规律,神经胶质细胞混合培养时小胶质细胞呈现阿米巴样,分离纯化3天后基本恢复分枝状;(2)RT-PCR结果表明,m AChRs五种亚型受体mRNA均在小胶质细胞表达,免疫荧光结合CLSM分析和Western-blot结果显示M1受体表达于小胶质细胞,并且定位在胞膜和胞浆;(3)免疫荧光结合CLSM分析发现,正常组小胶质细胞MHCΙ表达量较少,OX(10-5M)与小胶质细胞共同孵育72 h,MHCΙ表达显著上调(P0.001);(4)流式细胞术结果表明,正常组小胶质细胞膜共标记MHCΙ和β2m的细胞阳性率为8.46±0.47%,OX组β2m和MHCΙ共标记细胞阳性率为51.80±1.48%,与正常组比较,OX组显著增加了5倍(P0.001),表明OX促进小胶质细胞膜功能性MHCΙ表达;(5)荧光定量PCR结果发现,OX促进抗原递呈分子相关基因Rt1-Aw2、β2m和Tap1 mRNA表达(P0.001);(6)免疫荧光结合CLSM分析表明,OX(10-5M,40 min)同时上调小胶质细胞MHCΙ和M1受体表达(P0.001),MT7(10-8 M)预处理小胶质5 min,部分阻断OX诱导的MHCΙ和M1受体表达(P0.001);并且M1受体与MHCΙ存在共定位,提示M1受体参与OX上调MHCΙ表达;(7)OX动态影响小胶质细胞M1受体表达:CLSM结合Western-blot结果显示,OX(10-5M,72 h)上调M1受体表达(P0.01);荧光定量PCR结果表明,OX(10-5M,20 min)抑制M1受体mRNA表达,OX(40 min和72 h)促进M1受体mRNA表达,MT7部分阻断OX诱导的M1受体mRNA表达(P0.001);(8)CLSM分析和western-blot结果显示,OX(10-5M,72 h)上调小胶质细胞激活分子CD68和CD11b表达(P0.001,P0.01),表明OX促进小胶质细胞活化;MT7部分阻断OX对两者表达的诱导作用(P0.001);提示M1受体直接调控小胶质细胞活化。(9)OX(10-6 M,10-4 M)与小胶质细胞共同孵育24 h,小胶质细胞TNF-а和IL-10释放减少(P0.05,P0.01),OX对IL-1β释放无明显影响。结论:(1)成功改进了原代小胶质细胞培养方法,提高了小胶质细胞分离效率。(2)m AChRs五种亚型受体m RNA均在大鼠皮层小胶质细胞表达,M1受体表达在小胶质细胞胞膜和胞浆。(3)OX激动mAChRs,促进小胶质细胞抗原递呈功能,活化小胶质细胞并调控其炎性介质释放。(4)阻断M1受体,抑制OX诱导的MHCΙ表达和小胶质细胞活化,提示M1受体在mAChRs调控小胶质细胞免疫应答作用中发挥重要作用。
[Abstract]:Alzheimer 's disease (AD) is a chronic degenerative brain disease occurring in the aged and prophase, with progressive memory impairment, cognitive impairment, and personality change as clinical manifestation. In the aging society of population, AD has become the fourth major killer of cardiovascular and cerebrovascular diseases, tumors and stroke, threatening old age. Human health, reducing the quality of life, aggravating the family and social burden.AD causes complex and diverse, the specific pathogenesis is still unclear. In recent years, the study found that neuroinflammatory reaction is an important cause of AD in.AD patients with high activation of microglia (Microglia, MG), the increase of inflammatory factors. As a neurophlogistic The regulation of the immune function of MG has become a new target for the treatment of AD. The study of its immune function is also a hot spot in the field of neurodegenerative disease. The role of.MG in CNS is a double-edged sword, activated MG phagocyte residue, antigen presentation function, cytokine secretion participation in immune response, and removal of pathogens. However, excessive activation of MG causes chronic inflammation in CNS and aggravates brain damage. Therefore, strict regulation of MG immune response is the key to prevent the occurrence of neuritis. Acetylcholine is an important neurotransmitter involved in the learning, cognition, and memory of the central brain, and the receptors of acetylcholine include the muscarinine type choline receptor (Muscarinic acetylch). Oline receptors, mAChRs) and nicotinic choline receptor (Nicotinic acetylcholine receptor, nAChRs). Recent studies have shown that nAChRs plays a role in MG mediated neuritis in the acetylcholine system, and that the 7 nicotinic receptor (7nAChR) is expressed in MG and activates the inflammatory response. It is not clear to participate in the inflammatory regulation of MG. Earlier studies in this laboratory found that SD rats were intraperitoneally injected with m AChRs non selective antagonist, Trihexyphenidyl hydrochloride (THP), to promote MG proliferation and activation in the hippocampus and cortex, and up regulation of MG CD68 expression accompanied by M1 receptor expression, and both existed in common. Localization, suggesting that M receptor mediates the regulatory role of THP on MG activation. In this study, we will confirm the expression of mAChRs in MG for the first time, and observe the regulatory role of mAChRs on MG immune response. This study is beneficial to explore the relationship between M AChRs and neuritis, and develop a new mechanism for the development of AD, which is beneficial to search for the development of AD. The new target for the treatment of AD. We first established the culture of primary rat cortical microglia. In vitro, we confirmed the expression of mAChRs in microglia; using mAChRs non selective agonists to oxidize Oxotremorine (OX), mAChRs non selective antagonist and M1 receptor blocker of muscarinine toxin 7 (Muscarinic Toxin) 7, MT7) three kinds of tools, to study the regulatory role of mAChRs on microglia antigen presenting function, microglia activation and release of immuno inflammatory mediators. Methods: (1) the primary cultured rat cortical microglia culture method was established: cultured cortical neuroglia cells of SD rats born within 12 h for 8-9 days, and the stratification of microglia was obvious The number of microglia was separated and purified by trypsin mild digestion and shaking method. The purified microglia was basically recovered after 3 days and identified by microglia labeling molecule Iba1 immunofluorescence staining. (2) mAChRs was expressed in small colloid cells: using RT-PCR, Western-blot Technology MRNA and protein levels were used to detect the expression of mAChRs in microglia. (3) the effect of mAChRs on the function of microglia antigen presenting function: mAChRs non selective agonist OX treatment of microglia, laser confocal scanning microscope (Confocal laser scanning microscope, CLSM) combined with immunofluorescence, and flow cytometry to observe OX The effect of Major Histocompatibility Complex Complex (MHC) on the expression of molecular related gene Rt1-Aw2, beta 2M and Tap1 mRNA of microglia antigen was detected by OX. (4) M1 receptor involved in the antigenic action of microglia regulated by mAChRs Immunofluorescence combined with CLSM, Western-blot and fluorescence quantitative PCR to detect the changes in the expression of MHC molecules and M1 receptors of microglia. (5) the effect of mAChRs on the activation of microglia: immunofluorescence combined with CLSM and Western-blot detection OX alone, and MT7 and OX associated drugs on the activation of microglia and the expression of M1. (6) The effect of mAChRs on the release of microglia inflammatory mediators: Luminex technique was used to detect the effect of OX on the release of IL-1 beta, TNF- and IL-10 in microglia. Results: (1) the culture method of primary microglia was improved and the positive rate of Iba1 in microglia was up to 95%. The growth law of microglia and microglia were observed and microglia were cultured in mixed culture of glial cells. The cells presented amoeba like, after 3 days of isolation and purification, the branches were basically restored. (2) RT-PCR results showed that the five subtypes of M AChRs receptor mRNA were expressed in microglia, immunofluorescence combined with CLSM analysis and Western-blot results showed that the M1 receptor was expressed in the microglia and located in the cytoplasm and cytoplasm; (3) immunofluorescence combined with CLSM to analyze hair. The expression of MHC in normal microglia was less, OX (10-5M) incubated 72 h with microglia, and the expression of MHC was significantly up (P0.001). (4) flow cytometry results showed that the positive rate of MHC and beta 2m in normal group microglia membrane was 8.46 + 0.47%, and the positive rate of OX group beta 2M and MHC MHC was 51.80 + 1.48%. Compared with the normal group, the OX group increased by 5 times (P0.001), indicating that OX promoted the functional MHC expression of the microglia membrane; (5) the fluorescence quantitative PCR results found that OX promoted the antigen presenting molecular related genes Rt1-Aw2, beta 2M and Tap1 mRNA expression (P0.001); (6) immunofluorescence binding CLSM analysis indicated that the microglia was up regulated at the same time. M1 receptor expression (P0.001), MT7 (10-8 M) pretreated microglia 5 min, partially blocked OX induced MHC and M1 receptor expression (P0.001), and M1 receptor was Co located with MHC M1. Body expression (P0.01); fluorescence quantitative PCR results show that OX (10-5M, 20 min) inhibits the mRNA expression of M1 receptor, OX (40 min and 72 h) promotes M1 receptor mRNA expression. 1) showed that OX promoted the activation of microglia; MT7 partially blocked the induction of OX expression (P0.001); it suggested that M1 receptor directly regulate the activation of microglia. (9) OX (10-6 M, 10-4 M) incubated 24 h with microglia, TNF- and IL-10 release of microglia (P0.05,). Conclusion: (1) The primary microglia culture method was improved and the isolation efficiency of microglia was improved. (2) the expression of M AChRs subtype receptor m RNA in rat cortical microglia, M1 receptor expressed in the cell membrane and cytoplasm of microglia. (3) OX excitates mAChRs, promotes microglia antigen presenting function, activates microglia and regulates its inflammation The release of sexual media. (4) blocking the M1 receptor and inhibiting the expression of OX induced MHC and the activation of microglia, suggesting that the M1 receptor plays an important role in the regulation of the immune response of microglia by mAChRs.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16
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