缺血性脑卒中后固有免疫应答中小胶质细胞激活规律的年龄相关性差异

发布时间：2018-06-04 16:14

本文选题：小胶质细胞 + 神经炎症　；参考：《皖南医学院》2017年硕士论文

【摘要】：目的:炎症反应是脑缺血损伤后的基础病理生理过程,其中最关键的反应便是脑内固有免疫细胞的快速激活,主要是小胶质细胞(MG),它激活后的MG依据其作用不同分为M1经典激活型(促炎)及M2选择激活型(抗炎),所以说小胶质细胞是限制脑缺血后损伤的最新治疗靶点。尽管缺血性脑卒中的发生与年龄老化、炎症的增加有着密不可分的关系,但脑缺血发生后短时间内与年龄相关的病理机制尚不得而知,且老年大脑的生理生化状态与年轻大脑截然不同,对炎症的反应也极有可能不同。本文通过检测不同年龄脑缺血再灌注后M1/M2标志物表达水平及外周炎症介质表达水平,探讨缺血性脑卒中后年轻和老年大脑中小胶质细胞激活的规律及其差异性。方法:本课题采取建立大脑中动脉阻断模型(MCAO)模拟脑缺血损伤,随机选取健康雄性C57BL/6野生型(WT)小鼠3月龄和18月龄各只,相当于年轻人各自分为模型组和假手术组,模型组采用改良线栓法制备小鼠大脑中动脉阻塞再灌注局灶性脑缺血损伤模型,模型组缺血90min后,分别再灌注1天、3天和7天,每个时间点18只。记录每天神经功能缺损评分,再灌注结束后根据各个时间点取脑各6只分别行TTC染色测量梗死体积,免疫印迹(WB)测M1标志物iNOS及M2标志物Arg-1水平、免疫荧光染色分别共定位M1标志物MHCⅡ、M2标志物CD206水平和Iba1,共聚焦显微镜定位小胶质细胞,并分别计数,取外周血行酶联免疫吸附试验(ELISA)测定促炎因子TNF-α、i NOS、IL-4及抗炎因子IL-4、IL-10、TGF-β1表达水平明确系统炎性反应状况与神经炎症关系。最后使用单因素方差分析及独立样本T检验的方法分析老年大脑与年轻大脑的差异性。结果:1脑缺血再灌注1天、3天、7天后小鼠梗死体积:老年小鼠1天、3天、7天体积均小于年轻小鼠1天、3天、7天梗死体积,且各个时间点两两比较均有显著统计学差异(P0.01)。2神经功能缺损评分:与梗死体积比较结果相反,老年小鼠在1天、3天、7天神经功能缺损评分均高于年轻小鼠1天、3天、7天神经功能缺损评分,且各个时间点两两比较均有显著统计学差异(P0.01)。24h后的老年组和年轻组全部神经功能缺损结果同上。3 western blot测得老年组假手术组和3个时间点内表达Arg-1在第3天上升达到高峰后开始下降,而年轻组在第1天后便稳步上升,直到第7天大致顶峰。而年轻组各时间点表达水平明显高于老年组(P值均小于0.05老年组VS年轻组),且年轻组在sham、1天、3天、7天内呈现明显上升趋势,统计学具有差异性(P值均小于0.05年轻组sham.VS1day.VS.3.day.VS.7day)。脑缺血再灌注后抗炎因子逐步上升,。iNOS在老年组中为脑缺血再灌注后抗炎因子逐步上升,老年组M1水平较年轻组显著升高。4免疫荧光:假手术组中老年组与年轻组小胶质细胞的表达差异无统计学意义(老年sham.VS.年轻sham P0.05)。老年组在缺血再灌注3个时间点内,CD206的表达逐步上升至第7天开始下降,MHCⅡ在第1天即达到峰值,后逐渐下降。三个时间各数据变化具有统计学意义(老年组1天.VS.3天.VS.7天P0.05)。年轻组表达规律与老年组相似,但CD206的表达显著高于老年组,MHCⅡ的表达显著低于老年组(老年/年轻组1天.VS.老年/年轻组3天VS.老年/年轻组7天P0.05)。5 ELISA:外周血清中老年组假手术组促炎因子(TNF-α、INOS、IL-4)及抗炎因子(IL-4、IL-10、TGF-β1)水平无明显差异,而脑缺血再灌注后老年组机体释放显著高于年轻组机体的促炎因子及显著低于年轻组的抗炎因子。炎性因子较脑组织神经炎症迟滞。结论:我们实验研究结果表明:脑缺血损伤后,老年大脑与年轻大脑有着不同的炎症反应过程,M1的促炎作用主要发生在脑缺血再灌注后的前3天,而M2的抗炎作用发挥较晚,在3到7天。年轻大脑梗死体积比老年大脑拥有更大的梗死体积,但较轻的神经功能缺损,较低的死亡率和较好的预后,老年大脑外周促炎因子释放水平明显较年轻大脑高而抗炎因子释放水平较低。神经系统炎症可引发全身系统性炎症反应。
[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.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3

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