DAPK1在急性肺损伤炎症反应中的作用及机制研究

发布时间：2018-05-14 00:24

本文选题：急性肺损伤 + 死亡相关蛋白激酶1　；参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景:急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)是临床上较为常见的由创伤、严重感染等多种内外因素诱发的以难治性低氧血症为特征的危重症,目前认为ALI/ARDS的本质是一种炎症,其发病与炎症的失控密切相关。尽管近年来人们对ARDS的研究不断深入,诊疗技术有明显提升,其病死率仍居高不下[1-2]。因此,深入研究其发病机制,寻求新的有效的治疗靶点,开发新的药物和治疗手段,减轻过度炎症反应以降低发病率和死亡率显得尤为重要。死亡相关蛋白激酶1(DAPK1)是一种钙调蛋白(CaM)调节的丝氨酸/苏氨酸蛋白激酶,包含有多个功能区域,作为一种凋亡正性调节因子在多种途径诱导引起的凋亡中扮演着重要角色[3-4],还参与了炎症的调节和自噬[5-6]。有研究发现,DAPK1参与了脑及肾的缺血性急性损伤疾病过程[7-8]。然而,DAPK1是否也参与急性肺损伤的发病过程,是否参与其炎症失控的调节尚未见报道。因此,本课题拟采用脂多糖(LPS)构建急性肺损伤模型及LPS诱发的巨噬细胞炎症模型,观察DAPK1的表达变化,并通过下调DAPK1表达后观察细胞自噬水平、促炎基因表达及炎症因子表达水平,从而探讨DAPK1在急性肺损伤发病及炎症调节中的作用及机制。研究目的:1.明确DAPK1在急性肺损伤时的表达及其在炎症失控中的作用;2.探讨DAPK1在控制炎症反应过程中的潜在机制。方法:1.DAPK1在急性肺损伤小鼠肺组织中的表达研究通过LPS(10mg/kg)腹腔注射构建急性肺损伤小鼠模型,采取HE染色观察肺损伤病理改变情况,免疫组化、Western blot及Real-time PCR等技术明确DAPK1在急性肺损伤小鼠肺组织中的表达变化及定位,ELISA检测血浆促炎因子水平变化。2.DAPK1对LPS诱导小鼠巨噬细胞炎症反应的调控作用及机制研究Western blot及Real-time PCR技术观察LPS作用RAW264.7巨噬细胞后,DAPK1随时间和浓度改变的表达情况;构建DAPK1 shRNA腺相关病毒,感染巨噬细胞后应用Western blot观察对NK-KB启动子活性、下游p38-mTOR信号通路、自噬相关蛋白LC3、p62的影响,Real-time PCR检测炎症因子IL-6、TNF-α和IL-1β的表达变化。3.下调DAPK1的表达对急性肺损伤炎症反应的影响采取LPS腹腔注射构建急性肺损伤模型,使用DAPK1特异性抑制剂TC-DAPK6预处理小鼠抑制DAPK1表达,Western blot检测TC-DAPK6对DAPK1的抑制效果,HE染色观察DAPK1抑制后对LPS诱导肺损伤的病理改变情况,Western blot及Real-time PCR观察DAPK1抑制后对NF-κB P65及促炎因子水平的影响,应用Kaplan-Meier生存分析研究不同处理组小鼠的生存时间。结果:1.成功构建了急性肺损伤小鼠模型,DAPK1在正常小鼠肺组织中仅少量表达,在急性肺损伤3h时DAPK1表达较对照组升高(P0.05),至24h达到最高水平(P0.05)。炎症因子TNF-α、IL-6急性肺损伤时较对照组表达明显升高(P0.05),在12-24h维持在较高水平(P0.05)。随着LPS处理时间的延长,肺湿干重比逐渐升高(P0.05)。2.随着LPS浓度的不断升高,刺激RAW264.7巨噬细胞DAPK1表达逐渐增加(P0.05)。在LPS刺激6h后开始上升(P0.05),至24h达到最高水平(P0.05),至48h表达有所下降(P0.05)。此外,LPS处理RAW264.7细胞后,NF-κB启动子活性以及炎症因子IL-6、IL-1β、TNF-α的表达显著升高。通过AAV-DAPK1 shRNA转染下调巨噬细胞DAPK1表达后,NF-κB启动子活性明显下降(P0.05),同时P38-mTOR表达受到抑制,LC3 II/I上调,而p62表达减少(P0.05),自噬激活。此外,促炎因子IL-1β、IL-6、TNF-α表达水平也明显下降(P0.05)。3.TC-DAPK6特异性抑制DAPK1表达后,小鼠肺组织病理损伤较单独LPS组明显减轻,DAPK1表达受抑制后NF-κB p65表达明显下降(P0.05),血浆中促炎因子TNF-α、IL-6水平明显低于LPS组(p0.05),肺湿干重比显著降低。通过Kaplan-Meier生存分析发现,DAPK1抑制后小鼠的生存期显著长于LPS组(p0.01)。结论:1.DAPK1在LPS诱导的急性肺损伤中表达明显增加,可能参与急性肺损伤的发病。2.DAPK1通过调控P38-mTOR信号通路抑制巨噬细胞自噬以及激活NF-κB炎症信号通路,从而参与急性肺损伤的疾病过程。3.体内研究进一步证实DAPK1可通过激活NF-κB炎症信号通路,促进ARDS的发生发展。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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