本文選題：小膠質(zhì)細(xì)胞 + 免疫應(yīng)答　； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：阿爾茲海默癥(Alzheimer’s disease,AD)是發(fā)生在老年期及老年前期的一種慢性退化性腦變疾病,以進(jìn)行性記憶減退、認(rèn)知障礙、人格改變?yōu)榕R床表現(xiàn)。在人口老齡化的當(dāng)代社會(huì),AD成為繼心腦血管疾病,腫瘤和腦卒中的第四大殺手,威脅著老年人身體健康,降低了生活質(zhì)量,加重家庭和社會(huì)負(fù)擔(dān)。AD發(fā)病原因復(fù)雜多樣,目前具體發(fā)病機(jī)制仍不明確。近年研究發(fā)現(xiàn),神經(jīng)炎癥反應(yīng)是AD的一個(gè)重要的致病原因。AD患者腦內(nèi)小膠質(zhì)細(xì)胞(Microglia,MG)高度活化,炎性因子含量增高。作為神經(jīng)炎性的始作俑者,MG免疫功能的調(diào)控成為AD治療的新靶點(diǎn),其免疫功能的研究也躋身于神經(jīng)退行性疾病領(lǐng)域的研究熱點(diǎn)。MG在CNS中的作用是把雙刃劍,激活的MG吞噬細(xì)胞殘?jiān)?通過(guò)抗原遞呈功能、細(xì)胞因子分泌參與免疫應(yīng)答反應(yīng),移除病原體。然而,MG過(guò)度激活導(dǎo)致CNS發(fā)生慢性炎癥,加重大腦損傷。因此,嚴(yán)格調(diào)控MG免疫反應(yīng)是防止神經(jīng)炎性發(fā)生的關(guān)鍵。乙酰膽堿是一類參與中樞大腦學(xué)習(xí)、認(rèn)知、記憶等的重要神經(jīng)遞質(zhì),乙酰膽堿作用的受體包括毒蕈堿型膽堿受體(Muscarinic acetylcholine receptors,mAChRs)和煙堿型膽堿受體(Nicotinic acetylcholine receptor,nAChRs)。近年來(lái)研究表明,乙酰膽堿系統(tǒng)中nAChRs在MG介導(dǎo)的神經(jīng)炎性發(fā)揮作用,а7煙堿型受體(а7nAChR)在MG表達(dá),激活а7nAChR抑制MG的炎性反應(yīng)。然而,mAChRs在MG的表達(dá)情況以及是否參與MG的炎性調(diào)控尚不清楚。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),SD大鼠長(zhǎng)期腹腔注射m AChRs非選擇性拮抗劑鹽酸苯海索(Trihexyphenidyl hydrochloride,THP)促進(jìn)大腦海馬和皮層的MG增生和激活,上調(diào)MG的CD68表達(dá)同時(shí)伴隨M1受體表達(dá)增加,并且兩者存在共定位,暗示M受體介導(dǎo)THP對(duì)MG活化的調(diào)控作用。在本研究中,我們將首次確證mAChRs在MG的表達(dá)情況,并且觀察mAChRs對(duì)MG免疫應(yīng)答功能的調(diào)控作用。本研究的開(kāi)展利于探討m AChRs與神經(jīng)炎性的關(guān)系,開(kāi)拓了其影響AD發(fā)展的新機(jī)制研究,有利于尋找治療AD的新靶點(diǎn)。我們首先建立原代大鼠皮層小膠質(zhì)細(xì)胞培養(yǎng)方法,在體外條件下,確證mAChRs在小膠質(zhì)細(xì)胞表達(dá)情況;利用mAChRs非選擇性激動(dòng)劑氧化震顫素(Oxotremorine,OX)、mAChRs非選擇拮抗劑阿托品和M1受體阻斷劑毒蕈堿毒素7(Muscarinic Toxin 7,MT7)三種工具藥,研究mAChRs對(duì)小膠質(zhì)細(xì)胞抗原遞呈功能、小膠質(zhì)細(xì)胞活化和免疫炎癥介質(zhì)釋放的調(diào)控作用。方法:(1)建立大鼠皮層原代小膠質(zhì)細(xì)胞培養(yǎng)方法:將出生12 h內(nèi)的SD大鼠的皮層神經(jīng)膠質(zhì)細(xì)胞混合培養(yǎng)8-9天,小膠質(zhì)細(xì)胞分層明顯且數(shù)量達(dá)到最多,利用胰酶溫和消化法和震搖法進(jìn)行小膠質(zhì)細(xì)胞分離純化;純化的小膠質(zhì)細(xì)胞培養(yǎng)3天后基本恢復(fù)靜息狀態(tài),通過(guò)小膠質(zhì)細(xì)胞標(biāo)記分子Iba1免疫熒光染色進(jìn)行鑒定。(2)mAChRs在小膠質(zhì)細(xì)胞表達(dá)研究:利用RT-PCR、Western-blot技術(shù)從mRNA和蛋白水平檢測(cè)mAChRs在小膠質(zhì)細(xì)胞表達(dá)情況。(3)mAChRs對(duì)小膠質(zhì)細(xì)胞抗原遞呈功能影響研究:mAChRs非選擇性激動(dòng)劑OX處理小膠質(zhì)細(xì)胞,利用激光共聚焦掃描顯微鏡(Confocal laser scanning microscope,CLSM)結(jié)合免疫熒光、流式細(xì)胞術(shù)觀察OX對(duì)主要組織相容性抗原分子Ι(Major Histocompatibility ComplexΙ,MHCΙ)表達(dá)影響;熒光定量PCR檢測(cè)OX對(duì)小膠質(zhì)細(xì)胞抗原遞呈分子相關(guān)基因Rt1-Aw2、β2m和Tap1 mRNA表達(dá)影響。(4)M1受體參與mAChRs調(diào)控小膠質(zhì)細(xì)胞抗原遞呈作用研究:利用M1受體阻斷劑MT7,應(yīng)用免疫熒光結(jié)合CLSM、Western-blot和熒光定量PCR檢測(cè)MT7對(duì)小膠質(zhì)細(xì)胞MHCΙ分子和M1受體表達(dá)的變化。(5)mAChRs對(duì)小膠質(zhì)細(xì)胞活化的影響:免疫熒光結(jié)合CLSM和Western-blot檢測(cè)OX單獨(dú)給藥以及MT7和OX伴隨給藥對(duì)小膠質(zhì)細(xì)胞激活分子CD68和CD11b表達(dá)影響。(6)mAChRs對(duì)小膠質(zhì)細(xì)胞炎癥介質(zhì)釋放影響:Luminex技術(shù)檢測(cè)OX對(duì)小膠質(zhì)細(xì)胞IL-1β、TNF-а和IL-10釋放的影響。結(jié)果:(1)改進(jìn)了原代小膠質(zhì)細(xì)胞的培養(yǎng)方法,小膠質(zhì)細(xì)胞Iba1陽(yáng)性率高達(dá)95%;觀察了小膠質(zhì)細(xì)胞生長(zhǎng)規(guī)律,神經(jīng)膠質(zhì)細(xì)胞混合培養(yǎng)時(shí)小膠質(zhì)細(xì)胞呈現(xiàn)阿米巴樣,分離純化3天后基本恢復(fù)分枝狀;(2)RT-PCR結(jié)果表明,m AChRs五種亞型受體mRNA均在小膠質(zhì)細(xì)胞表達(dá),免疫熒光結(jié)合CLSM分析和Western-blot結(jié)果顯示M1受體表達(dá)于小膠質(zhì)細(xì)胞,并且定位在胞膜和胞漿;(3)免疫熒光結(jié)合CLSM分析發(fā)現(xiàn),正常組小膠質(zhì)細(xì)胞MHCΙ表達(dá)量較少,OX(10-5M)與小膠質(zhì)細(xì)胞共同孵育72 h,MHCΙ表達(dá)顯著上調(diào)(P0.001);(4)流式細(xì)胞術(shù)結(jié)果表明,正常組小膠質(zhì)細(xì)胞膜共標(biāo)記MHCΙ和β2m的細(xì)胞陽(yáng)性率為8.46±0.47%,OX組β2m和MHCΙ共標(biāo)記細(xì)胞陽(yáng)性率為51.80±1.48%,與正常組比較,OX組顯著增加了5倍(P0.001),表明OX促進(jìn)小膠質(zhì)細(xì)胞膜功能性MHCΙ表達(dá);(5)熒光定量PCR結(jié)果發(fā)現(xiàn),OX促進(jìn)抗原遞呈分子相關(guān)基因Rt1-Aw2、β2m和Tap1 mRNA表達(dá)(P0.001);(6)免疫熒光結(jié)合CLSM分析表明,OX(10-5M,40 min)同時(shí)上調(diào)小膠質(zhì)細(xì)胞MHCΙ和M1受體表達(dá)(P0.001),MT7(10-8 M)預(yù)處理小膠質(zhì)5 min,部分阻斷OX誘導(dǎo)的MHCΙ和M1受體表達(dá)(P0.001);并且M1受體與MHCΙ存在共定位,提示M1受體參與OX上調(diào)MHCΙ表達(dá);(7)OX動(dòng)態(tài)影響小膠質(zhì)細(xì)胞M1受體表達(dá):CLSM結(jié)合Western-blot結(jié)果顯示,OX(10-5M,72 h)上調(diào)M1受體表達(dá)(P0.01);熒光定量PCR結(jié)果表明,OX(10-5M,20 min)抑制M1受體mRNA表達(dá),OX(40 min和72 h)促進(jìn)M1受體mRNA表達(dá),MT7部分阻斷OX誘導(dǎo)的M1受體mRNA表達(dá)(P0.001);(8)CLSM分析和western-blot結(jié)果顯示,OX(10-5M,72 h)上調(diào)小膠質(zhì)細(xì)胞激活分子CD68和CD11b表達(dá)(P0.001,P0.01),表明OX促進(jìn)小膠質(zhì)細(xì)胞活化;MT7部分阻斷OX對(duì)兩者表達(dá)的誘導(dǎo)作用(P0.001);提示M1受體直接調(diào)控小膠質(zhì)細(xì)胞活化。(9)OX(10-6 M,10-4 M)與小膠質(zhì)細(xì)胞共同孵育24 h,小膠質(zhì)細(xì)胞TNF-а和IL-10釋放減少(P0.05,P0.01),OX對(duì)IL-1β釋放無(wú)明顯影響。結(jié)論:(1)成功改進(jìn)了原代小膠質(zhì)細(xì)胞培養(yǎng)方法,提高了小膠質(zhì)細(xì)胞分離效率。(2)m AChRs五種亞型受體m RNA均在大鼠皮層小膠質(zhì)細(xì)胞表達(dá),M1受體表達(dá)在小膠質(zhì)細(xì)胞胞膜和胞漿。(3)OX激動(dòng)mAChRs,促進(jìn)小膠質(zhì)細(xì)胞抗原遞呈功能,活化小膠質(zhì)細(xì)胞并調(diào)控其炎性介質(zhì)釋放。(4)阻斷M1受體,抑制OX誘導(dǎo)的MHCΙ表達(dá)和小膠質(zhì)細(xì)胞活化,提示M1受體在mAChRs調(diào)控小膠質(zhì)細(xì)胞免疫應(yīng)答作用中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16
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本文編號(hào)：2033252