本文選題：小膠質(zhì)細(xì)胞 + 神經(jīng)炎癥��； 參考：《皖南醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:炎癥反應(yīng)是腦缺血損傷后的基礎(chǔ)病理生理過程,其中最關(guān)鍵的反應(yīng)便是腦內(nèi)固有免疫細(xì)胞的快速激活,主要是小膠質(zhì)細(xì)胞(MG),它激活后的MG依據(jù)其作用不同分為M1經(jīng)典激活型(促炎)及M2選擇激活型(抗炎),所以說小膠質(zhì)細(xì)胞是限制腦缺血后損傷的最新治療靶點(diǎn)。盡管缺血性腦卒中的發(fā)生與年齡老化、炎癥的增加有著密不可分的關(guān)系,但腦缺血發(fā)生后短時(shí)間內(nèi)與年齡相關(guān)的病理機(jī)制尚不得而知,且老年大腦的生理生化狀態(tài)與年輕大腦截然不同,對(duì)炎癥的反應(yīng)也極有可能不同。本文通過檢測(cè)不同年齡腦缺血再灌注后M1/M2標(biāo)志物表達(dá)水平及外周炎癥介質(zhì)表達(dá)水平,探討缺血性腦卒中后年輕和老年大腦中小膠質(zhì)細(xì)胞激活的規(guī)律及其差異性。方法:本課題采取建立大腦中動(dòng)脈阻斷模型(MCAO)模擬腦缺血損傷,隨機(jī)選取健康雄性C57BL/6野生型(WT)小鼠3月齡和18月齡各只,相當(dāng)于年輕人各自分為模型組和假手術(shù)組,模型組采用改良線栓法制備小鼠大腦中動(dòng)脈阻塞再灌注局灶性腦缺血損傷模型,模型組缺血90min后,分別再灌注1天、3天和7天,每個(gè)時(shí)間點(diǎn)18只。記錄每天神經(jīng)功能缺損評(píng)分,再灌注結(jié)束后根據(jù)各個(gè)時(shí)間點(diǎn)取腦各6只分別行TTC染色測(cè)量梗死體積,免疫印跡(WB)測(cè)M1標(biāo)志物iNOS及M2標(biāo)志物Arg-1水平、免疫熒光染色分別共定位M1標(biāo)志物MHCⅡ、M2標(biāo)志物CD206水平和Iba1,共聚焦顯微鏡定位小膠質(zhì)細(xì)胞,并分別計(jì)數(shù),取外周血行酶聯(lián)免疫吸附試驗(yàn)(ELISA)測(cè)定促炎因子TNF-α、i NOS、IL-4及抗炎因子IL-4、IL-10、TGF-β1表達(dá)水平明確系統(tǒng)炎性反應(yīng)狀況與神經(jīng)炎癥關(guān)系。最后使用單因素方差分析及獨(dú)立樣本T檢驗(yàn)的方法分析老年大腦與年輕大腦的差異性。結(jié)果:1腦缺血再灌注1天、3天、7天后小鼠梗死體積:老年小鼠1天、3天、7天體積均小于年輕小鼠1天、3天、7天梗死體積,且各個(gè)時(shí)間點(diǎn)兩兩比較均有顯著統(tǒng)計(jì)學(xué)差異(P0.01)。2神經(jīng)功能缺損評(píng)分:與梗死體積比較結(jié)果相反,老年小鼠在1天、3天、7天神經(jīng)功能缺損評(píng)分均高于年輕小鼠1天、3天、7天神經(jīng)功能缺損評(píng)分,且各個(gè)時(shí)間點(diǎn)兩兩比較均有顯著統(tǒng)計(jì)學(xué)差異(P0.01)。24h后的老年組和年輕組全部神經(jīng)功能缺損結(jié)果同上。3 western blot測(cè)得老年組假手術(shù)組和3個(gè)時(shí)間點(diǎn)內(nèi)表達(dá)Arg-1在第3天上升達(dá)到高峰后開始下降,而年輕組在第1天后便穩(wěn)步上升,直到第7天大致頂峰。而年輕組各時(shí)間點(diǎn)表達(dá)水平明顯高于老年組(P值均小于0.05老年組VS年輕組),且年輕組在sham、1天、3天、7天內(nèi)呈現(xiàn)明顯上升趨勢(shì),統(tǒng)計(jì)學(xué)具有差異性(P值均小于0.05年輕組sham.VS1day.VS.3.day.VS.7day)。腦缺血再灌注后抗炎因子逐步上升,。iNOS在老年組中為腦缺血再灌注后抗炎因子逐步上升,老年組M1水平較年輕組顯著升高。4免疫熒光:假手術(shù)組中老年組與年輕組小膠質(zhì)細(xì)胞的表達(dá)差異無統(tǒng)計(jì)學(xué)意義(老年sham.VS.年輕sham P0.05)。老年組在缺血再灌注3個(gè)時(shí)間點(diǎn)內(nèi),CD206的表達(dá)逐步上升至第7天開始下降,MHCⅡ在第1天即達(dá)到峰值,后逐漸下降。三個(gè)時(shí)間各數(shù)據(jù)變化具有統(tǒng)計(jì)學(xué)意義(老年組1天.VS.3天.VS.7天P0.05)。年輕組表達(dá)規(guī)律與老年組相似,但CD206的表達(dá)顯著高于老年組,MHCⅡ的表達(dá)顯著低于老年組(老年/年輕組1天.VS.老年/年輕組3天VS.老年/年輕組7天P0.05)。5 ELISA:外周血清中老年組假手術(shù)組促炎因子(TNF-α、INOS、IL-4)及抗炎因子(IL-4、IL-10、TGF-β1)水平無明顯差異,而腦缺血再灌注后老年組機(jī)體釋放顯著高于年輕組機(jī)體的促炎因子及顯著低于年輕組的抗炎因子。炎性因子較腦組織神經(jīng)炎癥遲滯。結(jié)論:我們實(shí)驗(yàn)研究結(jié)果表明:腦缺血損傷后,老年大腦與年輕大腦有著不同的炎癥反應(yīng)過程,M1的促炎作用主要發(fā)生在腦缺血再灌注后的前3天,而M2的抗炎作用發(fā)揮較晚,在3到7天。年輕大腦梗死體積比老年大腦擁有更大的梗死體積,但較輕的神經(jīng)功能缺損,較低的死亡率和較好的預(yù)后,老年大腦外周促炎因子釋放水平明顯較年輕大腦高而抗炎因子釋放水平較低。神經(jīng)系統(tǒng)炎癥可引發(fā)全身系統(tǒng)性炎癥反應(yīng)。
[Abstract]:Objective: the inflammatory response is the basic pathophysiological process after cerebral ischemia. The most critical reaction is the rapid activation of the innate immune cells in the brain, mainly microglia (MG). The activated MG is divided into M1 classical activator (pro-inflammatory) and M2 selective activation type (anti-inflammatory), so the microglia is limited. Although the occurrence of ischemic stroke has an inseparable relationship with aging and the increase of inflammation, the pathological mechanism related to age is not known in a short time after the onset of cerebral ischemia, and the physiological and biochemical state of the brain is very different from that of the young brain, and the response to inflammation is extremely important. By detecting the expression level of M1/M2 markers and the expression level of peripheral inflammatory mediators after cerebral ischemia and reperfusion in different ages, the regularity and difference of the activation of medium and small glial cells in young and old brain after ischemic stroke are investigated. Methods: this subject adopts the establishment of a middle cerebral artery occlusion model (MCAO) to simulate brain deficiency. 3 month old and 18 month old healthy male C57BL/6 wild type (WT) mice were randomly selected and divided into model group and sham operation group. The model group was used to prepare the model of focal cerebral ischemia of middle cerebral artery occlusion and reperfusion in mice. After the ischemia 90min, the model group was reperfusion for 1 days, 3 days and 7 days, respectively. Each time point was 18. The scores of nerve function defect were recorded every day. After the reperfusion, the infarct volume was measured by TTC staining according to 6 brains at each time point. INOS and M2 marker Arg-1 level of M1 markers were measured by immunoblotting (WB). The M1 marker, MHC II, M2 marker CD206 level and Iba1, were confocal Microglia were located with microglia and were counted respectively. The expression of proinflammatory factor TNF- a, I NOS, IL-4 and anti-inflammatory factors IL-4, IL-10, TGF- beta 1 were determined by enzyme linked immunosorbent assay (ELISA), and the relationship between inflammatory reaction and neuroinflammation was clearly defined. Finally, the analysis of single factor analysis of variance and independent sample T test were used to analyze the age. The difference between the brain and the young brain. Results: 1 the infarct volume of mice in 1 days, 3 days and 7 days after cerebral ischemia and reperfusion: 1 days, 3 days and 7 days in aged mice were less than 1 days, 3 days, 7 days of infarct volume, and there were statistically significant differences (P0.01).2 neural function defect score: compared with infarct volume. On the contrary, the scores of nerve function defect of the aged mice were higher than that of young mice in 1 days, 3 days and 7 days, 3 days and 7 days of nerve function defect score, and all time points were statistically different (P0.01) after.24h, all the results of nerve function defect in the old group and the young group were same as.3 Western blot in the sham operation group and 3 of the elderly group. At the time point, the expression of Arg-1 began to fall after the rise in the third sky, and the young group rose steadily at first days until the seventh day, and the expression level of the young group was significantly higher than that of the old group (P value was less than the young group of VS in the 0.05 aged group), and the young group was in sham, 1 days, 3 days and 7 days, which showed an obvious upward trend. The study was different (P value was less than 0.05 younger than 0.05 young group sham.VS1day.VS.3.day.VS.7day). After cerebral ischemia and reperfusion, the anti inflammatory factors gradually increased. The anti inflammatory factors of.INOS in the elderly group increased gradually after cerebral ischemia-reperfusion. The M1 level of the elderly group was significantly higher than that of the younger group, and the.4 immunofluorescence was significantly increased in the elderly group: the elderly group in the sham operation group and the young group were microglia. The difference of cell expression was not statistically significant (aged sham.VS. young sham P0.05). In the 3 time points of ischemia reperfusion, the expression of CD206 gradually increased to seventh days, and MHC II reached its peak at the first day, then gradually decreased. The changes of each data had statistical significance at three times (1 days.VS.3 days P0.05 of the elderly group,.VS.7 days P0.05). The expression of light group was similar to that in the elderly group, but the expression of CD206 was significantly higher than that in the elderly group. The expression of MHC II was significantly lower than that in the elderly group (aged / young group,.VS. old / 1 days old / 3 days VS. old / young group 7 days P0.05).5 ELISA: in the elderly sham group (TNF- a, INOS, IL-4) and anti inflammatory factors (IL-4, IL-10, and beta 1). After cerebral ischemia and reperfusion, the body release of the old group was significantly higher than the young group's proinflammatory factor and significantly lower than the young group's anti-inflammatory factors. The inflammatory factor was more than the brain tissue neuro inflammatory delay. Conclusion: our experimental results show that the brain is different from the young brain after cerebral ischemia injury. The proinflammatory effect of M1 occurs mainly in the first 3 days after cerebral ischemia and reperfusion, and the anti-inflammatory effect of M2 is late, from 3 to 7 days. The infarct volume of the young brain is larger than that of the old brain, but the lighter nerve function defect, lower mortality and better prognosis, the release level of peripheral peripheral inflammation factor in the aged brain is clear. The higher the young brain and the lower the release level of anti-inflammatory factors. Nervous system inflammation can cause systemic systemic inflammatory response.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R743.3

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本文編號(hào)：1978013