本文關(guān)鍵詞： 膿毒癥相關(guān)性腦病 消退素D1 小膠質(zhì)細(xì)胞 促炎性細(xì)胞因子　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的最新膿毒癥(Sepsis)定義指出,膿毒癥是機(jī)體對(duì)感染的反應(yīng)失調(diào)而導(dǎo)致危及生命的器官功能障礙。大腦在膿毒癥的發(fā)展過程中起到至關(guān)重要的作用,不僅參與免疫調(diào)節(jié),而且也是膿毒癥病程進(jìn)展中的受累器官。膿毒癥相關(guān)性腦病(Sepsis-associated encephalopathy,SAE)是繼發(fā)于膿毒癥的彌漫性腦功能障礙及神經(jīng)系統(tǒng)臨床癥狀,患者通常不存在明顯的中樞神經(jīng)系統(tǒng)感染的情況。SAE臨床表現(xiàn)差異較大,輕者出現(xiàn)譫妄,重者則出現(xiàn)昏迷等癥狀。SAE患者死亡率高,ICU50%死亡患者與SAE相關(guān)。SAE引起的遠(yuǎn)期認(rèn)知功能改變是主要的神經(jīng)損傷之一,對(duì)患者生活質(zhì)量影響較大。既往研究發(fā)現(xiàn)胞外核苷酸、促炎性細(xì)胞因子、氧化應(yīng)激反應(yīng)可能是引起膿毒癥相關(guān)性腦病病理變化的主要原因。消退素(Resolvins)是近幾年研究較多的一種內(nèi)源性促炎癥消退脂質(zhì)介質(zhì),由ω-3脂肪酸二十二碳六烯酸(Docosahexaenoic acid,DHA)代謝產(chǎn)生消退素D1(Resolvin D1,Rv D1)在多種動(dòng)物模型中都體現(xiàn)出強(qiáng)大的促進(jìn)炎癥消退的作用,研究證實(shí)Rv D1能夠提高膿毒癥小鼠的生存率,減輕肺臟、腎臟等重要器官的炎性反應(yīng),起到保護(hù)和治療的作用。由此推測(cè)消退素D1能夠改善膿毒癥相關(guān)性腦病小鼠的預(yù)后,從而為膿毒癥相關(guān)性腦病患者的治療提供新的方向。本課題擬通過膿毒癥小鼠模型,研究Rv D1能否減少小膠質(zhì)細(xì)胞活化,降低促炎性細(xì)胞因子水平,減輕神經(jīng)系統(tǒng)炎癥反應(yīng),最終改善小鼠行為學(xué)的表現(xiàn),達(dá)到治療甚至治愈膿毒癥相關(guān)性腦病的目的。研究方法(一)小鼠膿毒癥相關(guān)性腦病模型的建立1、將20只C57BL/6小鼠隨機(jī)分成兩組,對(duì)照組(Control組,n=10)、盲腸結(jié)扎穿孔手術(shù)組(CLP組,n=10)。建模前每只小鼠進(jìn)行連續(xù)5天的水中運(yùn)動(dòng)訓(xùn)練。訓(xùn)練后次日CLP組行盲腸結(jié)扎穿孔術(shù)建立膿毒癥模型。術(shù)后第2天開始,兩組小鼠連續(xù)4天行隱蔽站臺(tái)實(shí)驗(yàn);術(shù)后第5天同時(shí)行空間探索實(shí)驗(yàn)。記錄兩組實(shí)驗(yàn)數(shù)據(jù)并統(tǒng)計(jì)分析相關(guān)指標(biāo)。2、將12只C57BL/6小鼠隨機(jī)分成兩組,對(duì)照組(Control組,n=6)、盲腸結(jié)扎穿孔手術(shù)組(CLP組,n=6)。24h后收集各組小鼠腦組織,觀察并比較兩組小鼠海馬組織IBA-1免疫組織化學(xué)染色并分析結(jié)果。(二)消退素D1對(duì)小鼠膿毒癥相關(guān)性腦病的保護(hù)作用1、將24只C57BL/6小鼠隨機(jī)分成四組,對(duì)照組(Control組,n=6)、盲腸結(jié)扎穿孔手術(shù)組(CLP組,n=6)、低劑量Rv D1處理組(CLP+Rv D1-L組,Rv D1 0.1μg/只,n=6)、高劑量Rv D1處理組(CLP+Rv D1-H組,Rv D1 1μg/只,n=6)。CLP造模后,即刻于小鼠尾靜脈注射對(duì)應(yīng)劑量Rv D1,CLP組只造模不給藥,24h后收集各組小鼠外周血血漿。檢測(cè)小鼠外周血促炎性細(xì)胞因子水平。2、將30只C57BL/6小鼠隨機(jī)分成三組,對(duì)照組(Control組,n=10)、盲腸結(jié)扎穿孔手術(shù)組(CLP組,n=10)、Rv D1處理組(CLP+Rv D1組,n=10)。建模前每只小鼠進(jìn)行連續(xù)5天的水迷宮訓(xùn)練。CLP+Rv D1組在術(shù)后即刻尾靜脈注射Rv D11μg/只,CLP組不給藥。術(shù)后第2天開始,連續(xù)4天行隱蔽站臺(tái)實(shí)驗(yàn);術(shù)后第5天同時(shí)行空間探索實(shí)驗(yàn)。記錄各組MWM數(shù)據(jù)并統(tǒng)計(jì)分析相關(guān)指標(biāo)。3、將18只C57BL/6小鼠隨機(jī)分成三組,對(duì)照組(Control組,n=6)、盲腸結(jié)扎穿孔手術(shù)組(CLP組,n=6)、Rv D1處理組(CLP+Rv D1組,Rv D1 1ug/只,n=6)。CLP造模后,即刻于小鼠尾靜脈注Rv D1 1μg/只,CLP組只造模不給藥,24h后收集各組小鼠腦組織,觀察并比較不同組小鼠海馬組織IBA-1免疫組織化學(xué)染色并分析結(jié)果。(三)消退素D1對(duì)小鼠膿毒癥相關(guān)性腦病治療作用的可能機(jī)制探索1、將24只C57BL/6小鼠隨機(jī)分成三組,對(duì)照組(Control組,n=8)、盲腸結(jié)扎穿孔手術(shù)組(CLP組,n=8)、Rv D1處理組(CLP+Rv D1組,Rv D1 1μg/只,n=8)。CLP造模后,即刻于小鼠尾靜脈注Rv D1,CLP組只造模不給藥,24h后收集各組小鼠外周血和海馬組織,檢測(cè)小鼠外周血促炎性細(xì)胞因子水平、海馬組織勻漿做PCR檢測(cè)相應(yīng)m RNA水平并分析結(jié)果。2、利用BV2細(xì)胞建立小膠質(zhì)細(xì)胞刺激模型,分為對(duì)照組(Control組),脂多糖(Lipopolysaccharide,LPS)刺激組(LPS組),Rv D1處理組(LPS+Rv D1組,Rv D1 10n M每孔)。檢測(cè)細(xì)胞上清促炎性細(xì)胞因子水平以及相應(yīng)m RNA水平并分析結(jié)果。結(jié)果(一)小鼠膿毒癥相關(guān)性腦病模型的建立1、在隱蔽站臺(tái)實(shí)驗(yàn)中,從術(shù)后第2天開始CLP組潛伏期略有縮短,無統(tǒng)計(jì)學(xué)意義;Control組從術(shù)后第2天開始潛伏期逐漸縮短,術(shù)后第5天(30.11±4.12)最短,與術(shù)后第2天(50.46±9.31)相比有統(tǒng)計(jì)學(xué)意義(P0.05);術(shù)后第5天潛伏期CLP組(47.42±9.23)較Control組(30.11±4.12)明顯延長(zhǎng)(P0.05)。在空間探索實(shí)驗(yàn)中,穿臺(tái)次數(shù)CLP組(0.65±0.40)較Control組(1.54±0.34)明顯降低(P0.05);目標(biāo)象限時(shí)間CLP組(28.01±6.70)較Control組(41.12±6.22)延長(zhǎng)(P0.05)2085。2、小鼠海馬組織IBA-1免疫組織化學(xué)染色結(jié)果,在×400倍數(shù)下觀察,CLP組小膠質(zhì)細(xì)胞活化數(shù)量明顯較Control組多,小膠質(zhì)細(xì)胞明顯胞體較大,核深染,細(xì)胞膜增殖伸出較多的突觸。(二)消退素D1對(duì)小鼠膿毒癥相關(guān)性腦病的保護(hù)作用1、CLP小鼠模型中,CLP+Rv D1-L組和CLP+Rv D1-H組IL-6、IL-1β、TNFα水平均較CLP組降低(P0.05)。且促炎性細(xì)胞因子,CLP+Rv D1-H組較CLP+Rv D1-L組水平低(P0.05),與濃度梯度呈相關(guān)性。后續(xù)實(shí)驗(yàn)可選用較高劑量作為干預(yù)劑量。2、在隱蔽站臺(tái)實(shí)驗(yàn)中,從術(shù)后第2天開始CLP組(51.36±9.02)與CLP+Rv D1組(51.78±9.17)和Control組(45.63±7.53)相比潛伏期延長(zhǎng)(P0.05);術(shù)后第3天,CLP+Rv D1組(40.83±6.37)較CLP組(43.25±5.37)潛伏期呈縮短趨勢(shì),與Control組(39.83±7.80)相比潛伏期仍延長(zhǎng);術(shù)后第4天開始,CLP+Rv D1組(32.57±19.86)較CLP組(45.75±5.48)潛伏期明顯縮短(P0.05),而與Control組(33.80±21.18)相比則無明顯差異。3、在空間探索實(shí)驗(yàn)中,各組穿臺(tái)率比較結(jié)果顯示,CLP組(0.88±0.14)vs Control組(1.52±0.24)(P0.05),CLP組(0.88±0.14)與CLP+Rv D1組(1.42±0.14)(P0.05),Rv D1治療組小鼠穿臺(tái)率較CLP組升高;各組目標(biāo)象限時(shí)間結(jié)果顯示,CLP組(26.85±4.06)與CLP+Rv D1組(18.17±1.76)較Control組(18.45±0.90)目標(biāo)象限時(shí)間延長(zhǎng),CLP組(26.85±4.06)vs CLP+Rv D1組(18.17±1.76)(P0.05),Rv D1治療組小鼠目標(biāo)象限時(shí)間較CLP組明顯延長(zhǎng)。4、小鼠海馬組織IBA-1免疫組織化學(xué)染色結(jié)果,在×200倍數(shù)下觀察,CLP組活化小膠質(zhì)細(xì)胞數(shù)量較Control組明顯增加(P0.05),同時(shí)CLP+Rv D1組較CLP組相比活化小膠質(zhì)細(xì)胞數(shù)量明顯減少(P0.05),但CLP+Rv D1組小膠質(zhì)細(xì)胞數(shù)相對(duì)多于Control組。CLP+Rv D1組較CLP組小膠質(zhì)細(xì)胞激活狀態(tài)表現(xiàn)不明顯,細(xì)胞胞體較CLP組小,細(xì)胞突觸較少。平均光密度結(jié)果提示,CLP+Rv D1組較CLP組平均光密度降低(P0.05),但較Control組稍高。(三)消退素D1對(duì)小鼠膿毒癥相關(guān)性腦病治療作用的可能機(jī)制探索1、在在體實(shí)驗(yàn)中,CLP+Rv D1組外周血IL-6、IL-1β、TNFα水平較CLP組明顯降低(P0.05)。相對(duì)應(yīng)的CLP+Rv D1組海馬組織中IL-6、IL-1β、TNFαm RNA表達(dá)量較CLP組也明顯減少(P0.05),提示消退素D1對(duì)SAE的保護(hù)作用不僅與減輕全身炎癥反應(yīng)有關(guān),同時(shí)還可能依賴于減輕腦內(nèi)的炎癥反應(yīng)。2、在體外BV2細(xì)胞實(shí)驗(yàn)中,LPS+Rv D1組細(xì)胞上清IL-6、IL-1β、TNFα分泌量較LPS組明顯降低(P0.05)。相對(duì)應(yīng)的LPS+Rv D1組BV2細(xì)胞中IL-6、IL-1β、TNFαm RNA表達(dá)量明顯減低(P0.05)。結(jié)論CLP術(shù)能夠穩(wěn)定的復(fù)制膿毒癥相關(guān)性腦病小鼠模型。消退素D1能夠從空間學(xué)習(xí)和空間記憶兩方面改善SAE小鼠認(rèn)知功能�？赡芘c消退素D1減輕全身炎癥反應(yīng)的同時(shí),減輕顱內(nèi)小膠質(zhì)細(xì)胞炎癥反應(yīng)水平有關(guān)。
[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 騫舵瘮杈冧袱緇勫皬榧犳搗椹粍緇嘔BA-1鍏嶇柅緇勭粐鍖栧鏌撹壊騫跺垎鏋愮粨鏋，

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