消退素D1对小鼠脓毒症相关性脑病的保护作用

发布时间：2018-01-24 23:42

  本文关键词： 脓毒症相关性脑病 消退素D1 小胶质细胞 促炎性细胞因子　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的最新脓毒症(Sepsis)定义指出,脓毒症是机体对感染的反应失调而导致危及生命的器官功能障碍。大脑在脓毒症的发展过程中起到至关重要的作用,不仅参与免疫调节,而且也是脓毒症病程进展中的受累器官。脓毒症相关性脑病(Sepsis-associated encephalopathy,SAE)是继发于脓毒症的弥漫性脑功能障碍及神经系统临床症状,患者通常不存在明显的中枢神经系统感染的情况。SAE临床表现差异较大,轻者出现谵妄,重者则出现昏迷等症状。SAE患者死亡率高,ICU50%死亡患者与SAE相关。SAE引起的远期认知功能改变是主要的神经损伤之一,对患者生活质量影响较大。既往研究发现胞外核苷酸、促炎性细胞因子、氧化应激反应可能是引起脓毒症相关性脑病病理变化的主要原因。消退素(Resolvins)是近几年研究较多的一种内源性促炎症消退脂质介质,由ω-3脂肪酸二十二碳六烯酸(Docosahexaenoic acid,DHA)代谢产生消退素D1(Resolvin D1,Rv D1)在多种动物模型中都体现出强大的促进炎症消退的作用,研究证实Rv D1能够提高脓毒症小鼠的生存率,减轻肺脏、肾脏等重要器官的炎性反应,起到保护和治疗的作用。由此推测消退素D1能够改善脓毒症相关性脑病小鼠的预后,从而为脓毒症相关性脑病患者的治疗提供新的方向。本课题拟通过脓毒症小鼠模型,研究Rv D1能否减少小胶质细胞活化,降低促炎性细胞因子水平,减轻神经系统炎症反应,最终改善小鼠行为学的表现,达到治疗甚至治愈脓毒症相关性脑病的目的。研究方法(一)小鼠脓毒症相关性脑病模型的建立1、将20只C57BL/6小鼠随机分成两组,对照组(Control组,n=10)、盲肠结扎穿孔手术组(CLP组,n=10)。建模前每只小鼠进行连续5天的水中运动训练。训练后次日CLP组行盲肠结扎穿孔术建立脓毒症模型。术后第2天开始,两组小鼠连续4天行隐蔽站台实验;术后第5天同时行空间探索实验。记录两组实验数据并统计分析相关指标。2、将12只C57BL/6小鼠随机分成两组,对照组(Control组,n=6)、盲肠结扎穿孔手术组(CLP组,n=6)。24h后收集各组小鼠脑组织,观察并比较两组小鼠海马组织IBA-1免疫组织化学染色并分析结果。(二)消退素D1对小鼠脓毒症相关性脑病的保护作用1、将24只C57BL/6小鼠随机分成四组,对照组(Control组,n=6)、盲肠结扎穿孔手术组(CLP组,n=6)、低剂量Rv D1处理组(CLP+Rv D1-L组,Rv D1 0.1μg/只,n=6)、高剂量Rv D1处理组(CLP+Rv D1-H组,Rv D1 1μg/只,n=6)。CLP造模后,即刻于小鼠尾静脉注射对应剂量Rv D1,CLP组只造模不给药,24h后收集各组小鼠外周血血浆。检测小鼠外周血促炎性细胞因子水平。2、将30只C57BL/6小鼠随机分成三组,对照组(Control组,n=10)、盲肠结扎穿孔手术组(CLP组,n=10)、Rv D1处理组(CLP+Rv D1组,n=10)。建模前每只小鼠进行连续5天的水迷宫训练。CLP+Rv D1组在术后即刻尾静脉注射Rv D11μg/只,CLP组不给药。术后第2天开始,连续4天行隐蔽站台实验;术后第5天同时行空间探索实验。记录各组MWM数据并统计分析相关指标。3、将18只C57BL/6小鼠随机分成三组,对照组(Control组,n=6)、盲肠结扎穿孔手术组(CLP组,n=6)、Rv D1处理组(CLP+Rv D1组,Rv D1 1ug/只,n=6)。CLP造模后,即刻于小鼠尾静脉注Rv D1 1μg/只,CLP组只造模不给药,24h后收集各组小鼠脑组织,观察并比较不同组小鼠海马组织IBA-1免疫组织化学染色并分析结果。(三)消退素D1对小鼠脓毒症相关性脑病治疗作用的可能机制探索1、将24只C57BL/6小鼠随机分成三组,对照组(Control组,n=8)、盲肠结扎穿孔手术组(CLP组,n=8)、Rv D1处理组(CLP+Rv D1组,Rv D1 1μg/只,n=8)。CLP造模后,即刻于小鼠尾静脉注Rv D1,CLP组只造模不给药,24h后收集各组小鼠外周血和海马组织,检测小鼠外周血促炎性细胞因子水平、海马组织匀浆做PCR检测相应m RNA水平并分析结果。2、利用BV2细胞建立小胶质细胞刺激模型,分为对照组(Control组),脂多糖(Lipopolysaccharide,LPS)刺激组(LPS组),Rv D1处理组(LPS+Rv D1组,Rv D1 10n M每孔)。检测细胞上清促炎性细胞因子水平以及相应m RNA水平并分析结果。结果(一)小鼠脓毒症相关性脑病模型的建立1、在隐蔽站台实验中,从术后第2天开始CLP组潜伏期略有缩短,无统计学意义;Control组从术后第2天开始潜伏期逐渐缩短,术后第5天(30.11±4.12)最短,与术后第2天(50.46±9.31)相比有统计学意义(P0.05);术后第5天潜伏期CLP组(47.42±9.23)较Control组(30.11±4.12)明显延长(P0.05)。在空间探索实验中,穿台次数CLP组(0.65±0.40)较Control组(1.54±0.34)明显降低(P0.05);目标象限时间CLP组(28.01±6.70)较Control组(41.12±6.22)延长(P0.05)2085。2、小鼠海马组织IBA-1免疫组织化学染色结果,在×400倍数下观察,CLP组小胶质细胞活化数量明显较Control组多,小胶质细胞明显胞体较大,核深染,细胞膜增殖伸出较多的突触。(二)消退素D1对小鼠脓毒症相关性脑病的保护作用1、CLP小鼠模型中,CLP+Rv D1-L组和CLP+Rv D1-H组IL-6、IL-1β、TNFα水平均较CLP组降低(P0.05)。且促炎性细胞因子,CLP+Rv D1-H组较CLP+Rv D1-L组水平低(P0.05),与浓度梯度呈相关性。后续实验可选用较高剂量作为干预剂量。2、在隐蔽站台实验中,从术后第2天开始CLP组(51.36±9.02)与CLP+Rv D1组(51.78±9.17)和Control组(45.63±7.53)相比潜伏期延长(P0.05);术后第3天,CLP+Rv D1组(40.83±6.37)较CLP组(43.25±5.37)潜伏期呈缩短趋势,与Control组(39.83±7.80)相比潜伏期仍延长;术后第4天开始,CLP+Rv D1组(32.57±19.86)较CLP组(45.75±5.48)潜伏期明显缩短(P0.05),而与Control组(33.80±21.18)相比则无明显差异。3、在空间探索实验中,各组穿台率比较结果显示,CLP组(0.88±0.14)vs Control组(1.52±0.24)(P0.05),CLP组(0.88±0.14)与CLP+Rv D1组(1.42±0.14)(P0.05),Rv D1治疗组小鼠穿台率较CLP组升高;各组目标象限时间结果显示,CLP组(26.85±4.06)与CLP+Rv D1组(18.17±1.76)较Control组(18.45±0.90)目标象限时间延长,CLP组(26.85±4.06)vs CLP+Rv D1组(18.17±1.76)(P0.05),Rv D1治疗组小鼠目标象限时间较CLP组明显延长。4、小鼠海马组织IBA-1免疫组织化学染色结果,在×200倍数下观察,CLP组活化小胶质细胞数量较Control组明显增加(P0.05),同时CLP+Rv D1组较CLP组相比活化小胶质细胞数量明显减少(P0.05),但CLP+Rv D1组小胶质细胞数相对多于Control组。CLP+Rv D1组较CLP组小胶质细胞激活状态表现不明显,细胞胞体较CLP组小,细胞突触较少。平均光密度结果提示,CLP+Rv D1组较CLP组平均光密度降低(P0.05),但较Control组稍高。(三)消退素D1对小鼠脓毒症相关性脑病治疗作用的可能机制探索1、在在体实验中,CLP+Rv D1组外周血IL-6、IL-1β、TNFα水平较CLP组明显降低(P0.05)。相对应的CLP+Rv D1组海马组织中IL-6、IL-1β、TNFαm RNA表达量较CLP组也明显减少(P0.05),提示消退素D1对SAE的保护作用不仅与减轻全身炎症反应有关,同时还可能依赖于减轻脑内的炎症反应。2、在体外BV2细胞实验中,LPS+Rv D1组细胞上清IL-6、IL-1β、TNFα分泌量较LPS组明显降低(P0.05)。相对应的LPS+Rv D1组BV2细胞中IL-6、IL-1β、TNFαm RNA表达量明显减低(P0.05)。结论CLP术能够稳定的复制脓毒症相关性脑病小鼠模型。消退素D1能够从空间学习和空间记忆两方面改善SAE小鼠认知功能。可能与消退素D1减轻全身炎症反应的同时,减轻颅内小胶质细胞炎症反应水平有关。
[Abstract]:Objective to study the new definition of sepsis (Sepsis) pointed out that sepsis is the body's response to infection disorders that lead to life-threatening organ dysfunction. The brain plays a crucial role in the development of sepsis, not only involved in immune regulation, but also the progress of sepsis in the course of sepsis affected organs. Sepsis associated encephalopathy (Sepsis-associated, encephalopathy, SAE) is secondary to sepsis with diffuse brain dysfunction and neurological symptoms in patients with central nervous system infection usually does not exist the obvious clinical manifestations of.SAE are quite different, the light emergence delirium, coma and other symptoms of severe cases of.SAE patients with high mortality, long-term death ICU50% the cognitive function of patients with SAE related changes caused by.SAE is one of the main nerve injury, a greater impact on the quality of life of the patients. Previous studies have found that extracellular nucleotides, proinflammatory Cytokines, oxidative stress may be the main cause of sepsis associated encephalopathy. The pathological changes of resolvin (Resolvins) in recent years is studied as an endogenous proinflammatory lipid mediators by regression, Omega -3 fatty acids with twenty-two carbon acid (Docosahexaenoic six acid, DHA) metabolism resolvin D1 (Resolvin D1, Rv, D1) in a variety of animal models are reflected in a powerful role in promoting inflammation subsided, the study confirmed that Rv D1 can improve the survival rate of sepsis mice to reduce lung inflammation, kidney and other organs, the protection and treatment effect. This can improve the prognosis of resolvin D1 sepsis sepsis associated encephalopathy in mice, so as to provide a new direction for the treatment of patients with sepsis associated encephalopathy. The aim of this mouse model of sepsis, Rv D1 can reduce microglial activation, reduce inflammatory The levels of cytokines, alleviate the inflammatory reaction of the nervous system, and ultimately improve the mice behavior performance, to treat or cure sepsis associated encephalopathy. Methods (1) to establish a mouse model of sepsis associated encephalopathy in 1, 20 C57BL/6 mice were randomly divided into two groups, control group (group Control, n=10), CLP Group (group CLP, n=10). All mice were in training for 5 consecutive days before modeling. Training the day after the CLP group underwent cecal ligation and puncture model of sepsis was established. Second days after operation, two groups of mice for 4 consecutive days the hidden platform test; fifth days after surgery at the same time for space exploration experiments. Records of two groups of experimental data and statistical analysis of the relevant indicators of.2, 12 C57BL/6 mice were randomly divided into two groups, control group (group Control, n=6), CLP Group (group CLP, n=6) collected in brain tissue of mice.24h after observation 骞舵瘮杈冧袱缁勫皬榧犳捣椹�缁勭粐IBA-1鍏嶇柅缁勭粐鍖栧�︽煋鑹插苟鍒嗘瀽缁撴灒�

本文编号：1461359