本文選題：急性肺損傷 + 死亡相關(guān)蛋白激酶1��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景:急性肺損傷(ALI)/急性呼吸窘迫綜合征(ARDS)是臨床上較為常見(jiàn)的由創(chuàng)傷、嚴(yán)重感染等多種內(nèi)外因素誘發(fā)的以難治性低氧血癥為特征的危重癥,目前認(rèn)為ALI/ARDS的本質(zhì)是一種炎癥,其發(fā)病與炎癥的失控密切相關(guān)。盡管近年來(lái)人們對(duì)ARDS的研究不斷深入,診療技術(shù)有明顯提升,其病死率仍居高不下[1-2]。因此,深入研究其發(fā)病機(jī)制,尋求新的有效的治療靶點(diǎn),開(kāi)發(fā)新的藥物和治療手段,減輕過(guò)度炎癥反應(yīng)以降低發(fā)病率和死亡率顯得尤為重要。死亡相關(guān)蛋白激酶1(DAPK1)是一種鈣調(diào)蛋白(CaM)調(diào)節(jié)的絲氨酸/蘇氨酸蛋白激酶,包含有多個(gè)功能區(qū)域,作為一種凋亡正性調(diào)節(jié)因子在多種途徑誘導(dǎo)引起的凋亡中扮演著重要角色[3-4],還參與了炎癥的調(diào)節(jié)和自噬[5-6]。有研究發(fā)現(xiàn),DAPK1參與了腦及腎的缺血性急性損傷疾病過(guò)程[7-8]。然而,DAPK1是否也參與急性肺損傷的發(fā)病過(guò)程,是否參與其炎癥失控的調(diào)節(jié)尚未見(jiàn)報(bào)道。因此,本課題擬采用脂多糖(LPS)構(gòu)建急性肺損傷模型及LPS誘發(fā)的巨噬細(xì)胞炎癥模型,觀察DAPK1的表達(dá)變化,并通過(guò)下調(diào)DAPK1表達(dá)后觀察細(xì)胞自噬水平、促炎基因表達(dá)及炎癥因子表達(dá)水平,從而探討DAPK1在急性肺損傷發(fā)病及炎癥調(diào)節(jié)中的作用及機(jī)制。研究目的:1.明確DAPK1在急性肺損傷時(shí)的表達(dá)及其在炎癥失控中的作用;2.探討DAPK1在控制炎癥反應(yīng)過(guò)程中的潛在機(jī)制。方法:1.DAPK1在急性肺損傷小鼠肺組織中的表達(dá)研究通過(guò)LPS(10mg/kg)腹腔注射構(gòu)建急性肺損傷小鼠模型,采取HE染色觀察肺損傷病理改變情況,免疫組化、Western blot及Real-time PCR等技術(shù)明確DAPK1在急性肺損傷小鼠肺組織中的表達(dá)變化及定位,ELISA檢測(cè)血漿促炎因子水平變化。2.DAPK1對(duì)LPS誘導(dǎo)小鼠巨噬細(xì)胞炎癥反應(yīng)的調(diào)控作用及機(jī)制研究Western blot及Real-time PCR技術(shù)觀察LPS作用RAW264.7巨噬細(xì)胞后,DAPK1隨時(shí)間和濃度改變的表達(dá)情況;構(gòu)建DAPK1 shRNA腺相關(guān)病毒,感染巨噬細(xì)胞后應(yīng)用Western blot觀察對(duì)NK-KB啟動(dòng)子活性、下游p38-mTOR信號(hào)通路、自噬相關(guān)蛋白LC3、p62的影響,Real-time PCR檢測(cè)炎癥因子IL-6、TNF-α和IL-1β的表達(dá)變化。3.下調(diào)DAPK1的表達(dá)對(duì)急性肺損傷炎癥反應(yīng)的影響采取LPS腹腔注射構(gòu)建急性肺損傷模型,使用DAPK1特異性抑制劑TC-DAPK6預(yù)處理小鼠抑制DAPK1表達(dá),Western blot檢測(cè)TC-DAPK6對(duì)DAPK1的抑制效果,HE染色觀察DAPK1抑制后對(duì)LPS誘導(dǎo)肺損傷的病理改變情況,Western blot及Real-time PCR觀察DAPK1抑制后對(duì)NF-κB P65及促炎因子水平的影響,應(yīng)用Kaplan-Meier生存分析研究不同處理組小鼠的生存時(shí)間。結(jié)果:1.成功構(gòu)建了急性肺損傷小鼠模型,DAPK1在正常小鼠肺組織中僅少量表達(dá),在急性肺損傷3h時(shí)DAPK1表達(dá)較對(duì)照組升高(P0.05),至24h達(dá)到最高水平(P0.05)。炎癥因子TNF-α、IL-6急性肺損傷時(shí)較對(duì)照組表達(dá)明顯升高(P0.05),在12-24h維持在較高水平(P0.05)。隨著LPS處理時(shí)間的延長(zhǎng),肺濕干重比逐漸升高(P0.05)。2.隨著LPS濃度的不斷升高,刺激RAW264.7巨噬細(xì)胞DAPK1表達(dá)逐漸增加(P0.05)。在LPS刺激6h后開(kāi)始上升(P0.05),至24h達(dá)到最高水平(P0.05),至48h表達(dá)有所下降(P0.05)。此外,LPS處理RAW264.7細(xì)胞后,NF-κB啟動(dòng)子活性以及炎癥因子IL-6、IL-1β、TNF-α的表達(dá)顯著升高。通過(guò)AAV-DAPK1 shRNA轉(zhuǎn)染下調(diào)巨噬細(xì)胞DAPK1表達(dá)后,NF-κB啟動(dòng)子活性明顯下降(P0.05),同時(shí)P38-mTOR表達(dá)受到抑制,LC3 II/I上調(diào),而p62表達(dá)減少(P0.05),自噬激活。此外,促炎因子IL-1β、IL-6、TNF-α表達(dá)水平也明顯下降(P0.05)。3.TC-DAPK6特異性抑制DAPK1表達(dá)后,小鼠肺組織病理?yè)p傷較單獨(dú)LPS組明顯減輕,DAPK1表達(dá)受抑制后NF-κB p65表達(dá)明顯下降(P0.05),血漿中促炎因子TNF-α、IL-6水平明顯低于LPS組(p0.05),肺濕干重比顯著降低。通過(guò)Kaplan-Meier生存分析發(fā)現(xiàn),DAPK1抑制后小鼠的生存期顯著長(zhǎng)于LPS組(p0.01)。結(jié)論:1.DAPK1在LPS誘導(dǎo)的急性肺損傷中表達(dá)明顯增加,可能參與急性肺損傷的發(fā)病。2.DAPK1通過(guò)調(diào)控P38-mTOR信號(hào)通路抑制巨噬細(xì)胞自噬以及激活NF-κB炎癥信號(hào)通路,從而參與急性肺損傷的疾病過(guò)程。3.體內(nèi)研究進(jìn)一步證實(shí)DAPK1可通過(guò)激活NF-κB炎癥信號(hào)通路,促進(jìn)ARDS的發(fā)生發(fā)展。
[Abstract]:Background: acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) is a clinically common critical disease characterized by multiple internal and external factors such as trauma and severe infection. It is considered that the essence of ALI/ARDS is a kind of inflammation, which is closely related to the loss of control of inflammation. The research of ARDS has been deepened, the diagnosis and treatment technology has been improved obviously and its mortality is still high, so it is still high [1-2].. Therefore, it is particularly important to study its pathogenesis, seek new effective therapeutic targets, develop new drugs and treatment methods, reduce the excessive inflammatory reaction in order to reduce the incidence and death rate. Death related protein kinase 1 (DAPK1) It is a serine / threonine protein kinase regulated by calmodulin (CaM), which contains multiple functional regions and plays an important role in apoptosis induced by multiple pathways as a positive regulator of apoptosis, [3-4]. It also participates in the regulation of inflammation and autophagic [5-6].. DAPK1 is involved in the acute ischemic loss of brain and kidney. It is not reported whether DAPK1 is involved in the pathogenesis of acute lung injury, however, whether it is involved in the regulation of acute lung injury, and it is not reported that it is not reported to participate in the regulation of inflammation. Therefore, we should use lipopolysaccharide (LPS) to construct acute lung injury model and LPS induced macrophage inflammation model, observe the expression changes of DAPK1, and observe the expression of DAPK1 by decreasing the expression of DAPK1. The level of autophagy, proinflammatory gene expression and expression of inflammatory factors in order to explore the role and mechanism of DAPK1 in the pathogenesis and regulation of acute lung injury. 1. the purpose of this study is to clarify the expression of DAPK1 in acute lung injury and its role in the control of inflammation, and 2. to explore the potential mechanism of DAPK1 in the process of controlling the inflammatory response. The expression of 1.DAPK1 in lung tissue of mice with acute lung injury was studied by intraperitoneal injection of LPS (10mg/kg) to construct a mouse model of acute lung injury. The pathological changes of lung injury were observed by HE staining, immunohistochemistry, Western blot and Real-time PCR were used to clarify the expression and localization of DAPK1 in the lung tissue of mice with acute lung injury, ELISA. The regulatory role of plasma proinflammatory factor level changes.2.DAPK1 on LPS induced macrophage inflammatory response and its mechanism study Western blot and Real-time PCR technology to observe the expression of DAPK1 with time and concentration after LPS action RAW264.7 macrophages; construct DAPK1 shRNA gland associated virus, infected macrophages and apply Weste The effect of RN blot on NK-KB promoter activity, downstream p38-mTOR signaling pathway, the effect of autophagy related protein LC3, p62, Real-time PCR detection of the expression of inflammatory factors IL-6, TNF- alpha and IL-1 beta, the expression of TNF- A and IL-1 beta has an effect on acute lung injury by constructing an acute lung injury model by intraperitoneal injection. The preparation TC-DAPK6 pretreated mice to inhibit the expression of DAPK1, Western blot was used to detect the inhibitory effect of TC-DAPK6 on DAPK1. The pathological changes of LPS induced lung injury after DAPK1 inhibition were observed by HE staining. The effect of Western blot on the inhibition of DAPK1 and the level of proinflammatory factors after inhibition of Western blot were studied. The survival time of the mice in the same treatment group. Results: 1. a mouse model of acute lung injury was successfully constructed. Only a small amount of DAPK1 was expressed in the lung tissue of normal mice. The expression of DAPK1 in the acute lung injury was higher than that in the control group (P0.05), to the highest level (P0.05) to 24h (P0.05). The expression of inflammatory factor TNF- A and IL-6 in acute lung injury were significantly higher than those in the control group (P). 0.05) at the high level of 12-24h (P0.05). With the prolongation of LPS treatment time, the ratio of wet dry weight of lung gradually increased (P0.05).2. with the increase of LPS concentration, and the expression of DAPK1 in RAW264.7 macrophages increased gradually (P0.05). In addition, after LPS treatment of RAW264.7 cells, the activity of NF- kappa B promoter and the expression of IL-6, IL-1 beta, and TNF- alpha were significantly increased. The activity of NF- kappa B promoter was significantly decreased after AAV-DAPK1 shRNA transfection, and the expression was inhibited and autophagy stimulated. In addition, the expression level of IL-1 beta, IL-6, TNF- alpha was also significantly decreased (P0.05).3.TC-DAPK6 specific inhibition of DAPK1 expression, the pathological damage of lung tissue in mice was significantly lower than that in the single LPS group, and the p65 expression of NF- kappa B decreased significantly after the inhibition of DAPK1 expression (P0.05), and the level of proinflammatory factor alpha in the plasma was significantly lower than that of the lung. The Kaplan-Meier survival analysis showed that the survival time of DAPK1 inhibited mice was significantly longer than that of the LPS group (P0.01). Conclusion: 1.DAPK1 was significantly increased in the acute lung injury induced by LPS, and may be involved in the pathogenesis of acute lung injury by regulating the autophagy of macrophages and activating N by regulating the P38-mTOR signaling pathway. The F- kappa B inflammatory signaling pathway, which is involved in the disease process of acute lung injury, has further confirmed that DAPK1 can promote the development of ARDS by activating the NF- kappa B signaling pathway.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R563.8

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3 孫建斌;雙鏈核糖核酸（dsRNA）的產(chǎn)生及其調(diào)控的信號(hào)通路在急性肺損傷發(fā)生發(fā)展中的分子機(jī)制研究[D];第四軍醫(yī)大學(xué);2015年

4 蘇玉杰;保肺解窘合劑治療膿毒癥致急性肺損傷的臨床研究[D];成都中醫(yī)藥大學(xué);2015年

5 張勇;褪黑素通過(guò)抑制NLRP3炎性小體減輕小鼠急性肺損傷[D];中國(guó)農(nóng)業(yè)大學(xué);2016年

6 祝華;臍帶間充質(zhì)干細(xì)胞治療急性肺損傷模型的機(jī)制探討[D];重慶醫(yī)科大學(xué);2015年

7 賈春娥;陰離子交換轉(zhuǎn)運(yùn)蛋白Pendrin在急性肺損傷小鼠模型中的作用和干預(yù)研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

8 杜松林;α1-抗胰蛋白酶對(duì)體外循環(huán)術(shù)后急性肺損傷的保護(hù)作用研究[D];南方醫(yī)科大學(xué);2016年

9 王勤;解偶聯(lián)蛋白2在脂多糖誘導(dǎo)的急性肺損傷中的作用及機(jī)制研究[D];第三軍醫(yī)大學(xué);2015年

10 趙燕;雷帕霉素通過(guò)抑制Th17細(xì)胞分化減輕LPS誘導(dǎo)的小鼠急性肺損傷[D];重慶醫(yī)科大學(xué);2016年

相關(guān)碩士學(xué)位論文 前10條

1 劉霞;DAPK1在急性肺損傷炎癥反應(yīng)中的作用及機(jī)制研究[D];第三軍醫(yī)大學(xué);2017年

2 劉安;間充質(zhì)干細(xì)胞在急性肺損傷/急性呼吸窘迫綜合征中的研究進(jìn)展[D];蚌埠醫(yī)學(xué)院;2013年

3 何鑫敏;氦氧通氣對(duì)急性肺損傷病人的治療作用及機(jī)制[D];福建醫(yī)科大學(xué);2015年

4 任鴻昌;MMP-9、AQP-1、AQP-5在胰蛋白酶導(dǎo)致的急性肺損傷中的表達(dá)及意義[D];安徽醫(yī)科大學(xué);2015年

5 宣國(guó)平;Ang2在急性肺損傷中的作用及其機(jī)制研究[D];安徽醫(yī)科大學(xué);2015年

6 侯林義;抗CD14單克隆抗體對(duì)膿毒癥急性肺損傷大鼠肺泡巨噬細(xì)胞內(nèi)NF-κB(p65)影響的研究[D];山西醫(yī)科大學(xué);2015年

7 馮迪;PPAR γ通路參與電針預(yù)處理對(duì)急性肺損傷的保護(hù)作用及相關(guān)機(jī)制[D];蘇州大學(xué);2015年

8 焦艷;PGC-1α在急性肺損傷中的作用及機(jī)制研究[D];第三軍醫(yī)大學(xué);2015年

9 莊佩佩;LPS誘導(dǎo)小鼠急性肺損傷及MgSO_4對(duì)其保護(hù)作用機(jī)制的研究[D];寧夏大學(xué);2015年

10 江偉哲;右美托咪定對(duì)大鼠急性肺損傷時(shí)肺組織NF-κB活性及細(xì)胞因子的影響[D];四川醫(yī)科大學(xué);2015年

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