SIRT1对LPS致急性呼吸窘迫综合征的炎症作用及机制研究

发布时间：2018-06-09 20:14

本文选题：SIRT1 + 急性呼吸窘迫综合征　；参考：《第三军医大学》2017年硕士论文

【摘要】：背景与目的:ARDS是以严重呼吸窘迫,进行性呼吸衰竭为主要表现的临床急、危重症[1]。其病理特征为肺泡上皮屏障和肺毛细血管内皮屏障的弥漫性损害,导致蛋白质渗漏、肺水肿、炎性细胞因子浸润及透明膜形成等[1、6]。直接或间接的肺损伤均引起炎症细胞的活化及炎症介质的大量释放,继而触发炎症级联反应,导致全身炎症失控[4]。临床上ARDS患者即使有相似的发病因素、治疗方案,其发展为ARDS的概率,炎症程度,进展情况及预后也存在差异。因此深入研究与ARDS的炎症发生密切相关的基因将对ARDS的防治提供新的方向。SIRT1是Ⅲ类组蛋白去乙酰化酶(histone deacetylase,HDAC),近来研究证实,SIRT1可通过对NF-κB、FOXO3、p53等组蛋白和非组蛋白的脱乙酰基作用,启动炎症的发生发展[7、11]。但SIRT1对炎症的作用是双向的,SIRT1可以抑制炎症因子的活化,持续增高的水平又会出现免疫抑制并造成死亡率增加,SIRT1对炎症的作用可因环境的不同而不同[15-17]。疾病阶段、作用靶点的差异等,SIRT1对机体的炎症调控及疾病转归可能都存在差异。而SIRT1对ARDS的炎症反应究竟有着何种作用尚不清楚,而p38 MAPK信号通路作为ARDS的主要炎症调控途径,因此推测p38 MAPK信号通路可能参与SIRT1对ARDS的炎症作用。因此本课题拟运用SIRT1+/-基因敲减小鼠和野生型小鼠,在不同SIRT1基因背景下,用LPS建立ARDS模型,评估SIRT1基因减弱对肺损伤炎症程度的影响,以及观察肺组织中p38 MAPK通路信号分子表达的变化,从而初步探讨SIRT1对LPS致ARDS炎症发生发展的具体作用及其可能机制。方法:1.本研究采用SIRT1+/-基因敲减小鼠作为实验动物,进行大量配对繁殖,用PCR法进行基因型鉴定,筛选出足够数量的SIRT1+/-小鼠,用于后续实验研究。2.运用SIRT1+/-基因敲减和野生型小鼠,使用q RT-PCR和Western Blot检测两种小鼠肺组织中SIRT1表达的差异。3.用LPS腹腔注射法建立ARDS模型,观察两种不同SIRT1基因背景小鼠的肺组织病理形态学变化,测定肺湿干比(W/D比),进行支气管肺泡灌洗液(BALF)的白细胞计数,BCA法测定BALF中总蛋白浓度,ELISA法检测BALF及血浆中炎症因子TNF-ɑ、IL-6水平,Western Blot及免疫组化法检测两种小鼠肺组织中p-p38MAPK、p-ATF2的表达变化。结果:1.SIRT1+/-基因敲减杂合子小鼠(HET)与野生型小鼠(WT)相比,其肺组织SIRT1在m RNA和蛋白表达均显著降低(P0.01)。2.HET+LPS组与WT+LPS组分别与各自生理盐水对照组相比,病理形态学观察均表现为肺组织的明显损伤,肺损伤病理评分显著增高(P0.05),而HET+LPS组中肺组织结构的破坏更严重,肺损伤病理评分进一步增加(P0.05);HET+LPS组与WT+LPS组相比于对照组,肺W/D比,BALF中白细胞总数、总蛋白浓度,BALF及血浆中炎症因子TNF-ɑ、IL-6水平均显著增加(P0.05),而HET+LPS组与WT+LPS组相比,上述指标的增加更为显著(P0.05)。3.HET+LPS组与WT+LPS组分别和各自对照组相比,肺组织中p-p38MAPK、p-ATF2的表达增加(P0.05),而HET+LPS组与WT+LPS组相比,上述蛋白的增加更明显(P0.05)。结论:1.SIRT1+/-小鼠肺组织中的SIRT1表达显著降低,SIRT1表达的差异为后续实验提供了必要的前提和有效的保障。2.LPS致ARDS的疾病模型中,SIRT1+/-小鼠与野生型小鼠相比,表现为更严重的炎症反应,提示SIRT1敲减对LPS致ARDS小鼠有着促炎作用,SIRT1在LPS致ARDS的炎症反应过程中起着保护作用。3.LPS致ARDS的疾病模型中,SIRT1+/-小鼠肺组织中p-p38MAPK、p-ATF2的表达与野生型小鼠相比明显增加,提示p38 MAPK-p-ATF2信号通路可能参与了SIRT1对LPS致ARDS的炎症调控作用。
[Abstract]:Background and purpose: ARDS is an acute clinical manifestation of severe respiratory distress and progressive respiratory failure. The pathological features of critical [1]. are diffuse impairment of the alveolar epithelial barrier and the barrier of pulmonary capillary endothelium, resulting in protein leakage, pulmonary edema, inflammatory cytokines infiltration, and the formation of transparent membrane and other direct or indirect lung lesions, such as [1,6].. The injury causes the activation of inflammatory cells and the massive release of the inflammatory mediators, and then triggers the inflammatory cascade reaction, which leads to the [4]. patients with systemic inflammation, even if there are similar pathogenesis, treatment schemes, the probability of developing ARDS, the degree of inflammation, progress and preconditioning. Therefore, the study and the inflammation of the ARDS are deeply studied. Closely related genes will provide a new direction for the prevention and control of ARDS..SIRT1 is class III histone deacetylase (histone deacetylase, HDAC). Recent studies have confirmed that SIRT1 can initiate the occurrence and development of inflammation by the deacetylation of NF- kappa B, FOXO3, p53 and other histones, but the effect of SIRT1 on inflammation is bidirectional. SIRT1 can inhibit the activation of inflammatory factors, the level of sustained increased levels of immunosuppression and the increase in mortality, the effect of SIRT1 on inflammation can be different in the [15-17]. disease stage, the difference of target target, and the difference in the regulation of inflammation and the outcome of the disease by SIRT1, and SIRT1 to ARDS What is the role of the disease response is not clear, and the p38 MAPK signaling pathway is the main regulation of inflammation in ARDS, so it is presumed that the p38 MAPK signaling pathway may be involved in the inflammatory effect of SIRT1 on ARDS. Therefore, we should use SIRT1+/- knockout mice and wild mice to establish ARDS mode with LPS in different SIRT1 background. To assess the effect of SIRT1 gene weakening on the degree of inflammation in lung injury, and to observe the changes in the expression of signal molecules in the p38 MAPK pathway in lung tissue, and to explore the specific role and possible mechanism of SIRT1 on the development of LPS induced ARDS inflammation. Methods: 1. studies were conducted with SIRT1+/- gene knockout mice as experimental animals. For reproduction, PCR method was used to identify the genotypes and to screen out a sufficient number of SIRT1+/- mice. For the follow-up experiment,.2. was used to use SIRT1+/- gene knockout and wild type mice. Q RT-PCR and Western Blot were used to detect the difference of SIRT1 expression in the lung tissues of two mice..3. LPS abdominal injection was used to establish ARDS model, and two different kinds of bases were observed. The pulmonary wetness and dry ratio (W/D ratio), the white cell count of bronchoalveolar lavage fluid (BALF), the total protein concentration in BALF were measured by the BCA method. The ELISA method was used to detect the BALF and the inflammatory factor TNF- in the plasma, the IL-6 level, Western Blot and immunohistochemistry were used to detect the p-p38MAPK in the lung tissues of two mice. P-A Results: the expression of TF2 in the 1.SIRT1+/- gene knockout heterozygote mice (HET) and the wild type mice (WT), the expression of SIRT1 in the lung tissue was significantly decreased (P0.01) in the m RNA and protein (P0.01) in the.2.HET+LPS group and the WT+LPS group, compared with the normal saline control group, the pathological morphologic observation all showed the obvious injury of the lung tissue and the lung injury disease. The lung tissue structure in HET+LPS group was significantly increased (P0.05), but the damage of lung tissue structure was more serious and the pathological score of lung injury was further increased (P0.05). Compared with the control group, the HET+LPS group was compared with the control group, the W/D ratio in the lung, the total white blood cells in the BALF, the total protein concentration, the BALF and the IL-6 water increased significantly (P0.05), while the HET+LPS group was with the WT+LPS. Compared with the +LPS group, the above index increased significantly (P0.05) the expression of p-p38MAPK and p-ATF2 increased in the lung tissue compared with the WT+LPS group and the control group (P0.05), while the increase of the above protein was more obvious in the group HET+LPS than in the WT+LPS group (P0.05). Conclusion: the SIRT1 expression in the lung tissue of the 1.SIRT1+/- mice decreased significantly. The difference in expression provides the necessary premise for the follow-up experiment and the effective guarantee of the disease model of.2.LPS induced ARDS. The SIRT1+/- mice are more severe inflammatory response compared with the wild type mice, suggesting that the SIRT1 knockout has the proinflammatory effect on the ARDS mice induced by LPS, and the SIRT1 plays a protective role in the process of ARDS's inflammation induced by LPS.3.LPS. In the ARDS disease model, the expression of p-p38MAPK and p-ATF2 in the lung tissue of SIRT1+/- mice increased significantly compared with the wild type mice, suggesting that the p38 MAPK-p-ATF2 signaling pathway may be involved in the regulation of SIRT1 to LPS induced ARDS.
【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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