外源性腺苷对脂多糖刺激的中性粒细胞趋化功能的调控作用及分子机制

发布时间：2018-05-29 01:21

本文选题：脓毒症 + 中性粒细胞　；参考：《江苏大学》2017年硕士论文

【摘要】：背景与目的脓毒症是宿主对微生物感染的反应失调导致了危及生命的器官功能障碍,是感染、烧/创伤、休克等急危重患者的严重并发症。随着当代医疗技术、危重病监护手段、治疗措施的进步,但其病死率依然居高不下。脓毒症时,宿主免疫功能严重失调,机体自身清除感染能力下降,是导致感染难以控制和多器官功能障碍的重要原因。中性粒细胞作为防御病原体的第一道防线,其依赖趋化功能来发挥杀灭细菌作用。脓毒症时,在多种细菌毒素及细胞因子的刺激下,中性粒细胞功能失调,其招募和趋化功能严重受损,吞噬能力下降,炎症介质释放增多,NETs形成增加,是脓毒症患者免疫功能紊乱的重要原因。因此,如何调控脓毒症患者自身的免疫功能,从而有效的发挥抗感染的作用,可能成为治疗脓毒症的一个重要手段之一。脂多糖(LPS)是一种高度酰化的糖脂,位于革兰氏阴性(G-)杆菌外膜的外层,是G-杆菌感染所致的内毒素血症及脓毒症时最重要的致病成分。早期在体和体外研究均表明LPS能够抑制中性粒细胞的趋化功能。腺苷(ADO)是腺嘌呤核苷三磷酸(ATP)的水解产物之一,半衰期很短,但具有强大的效应功能。ADO发挥其功能是通过4种G蛋白偶联受体:A1、A2A、A2B和A3受体。这4种ADO受体均在中性粒细胞表面表达,ADO可通过其不同的受体介导中性粒细胞多种功能包括调控中性粒细胞的趋化功能,而关于ADO介导的恢复LPS抑制的中性粒细胞趋化作用目前尚无报道。方法1.采集健康成年志愿者肘部静脉血,通过Ficoll密度梯度离心法分离健康成人外周血中性粒细胞;采用瑞士染色、台盼蓝染色及流式细胞术检测CD66b,鉴定中性粒细胞的活性及纯度。2.建立琼脂糖下趋化模型,使用趋化物IL-8和f MLP检测中性粒细胞的定向迁移距离,鉴定趋化模型及确立趋化物的使用方案。3.对中性粒细胞进行LPS刺激、ADO干预、ADO受体相关抑制剂和激动剂、c AMP类似物和抑制剂、AC激动剂、p38 MAPK抑制剂等干预,采用琼脂糖下趋化模型检测不同干预下中性粒细胞的定向迁移能力。4.采用流式细胞术和免疫印记(WB)检测中性粒细胞ADO受体A1的表达。5.采用蛋白芯片检测MAPK信号通路相关蛋白的磷酸化水平;WB检测总p38 MAPK相对磷酸化水平。结果1.Ficoll密度梯度离心法分离的细胞,瑞士染色发现细胞核染色为紫色,呈现马蹄形或分叶状,细胞胞质呈现浅红色,为中性粒细胞;台盼蓝染色及流式细胞术检测CD66b,分别显示细胞的活性和纯度均≥97%。2.琼脂糖下趋化实验显示IL-8和f MLP诱导中性粒细胞趋化的最适浓度分别为1μmol/L和0.1μmol/L。3.LPS刺激中性粒细胞后,可以明显抑制中性粒细胞定向迁移到IL-8或f MLP,呈剂量依赖方式;ADO干预能够恢复LPS抑制的中性粒细胞的趋化,呈浓度依赖方式。4.ADO介导的恢复LPS抑制的中性粒细胞趋化主要是通过中性粒细胞表面的ADO受体A1发挥作用。5.LPS刺激中性粒细胞后,其对A1受体(A1R)表达水平未见明显改变。6.A1R发挥恢复LPS抑制的中性粒细胞趋化作用可能不依赖其经典的AC-c AMP-PKA信号通路,而是抑制p38 MAPK磷酸化水平。
[Abstract]:Background and objective sepsis is the disorder of the host's response to microbial infection, which leads to life-threatening organ dysfunction, infection, fever, trauma, shock and other severe complications. With the development of modern medical technology, critical care and treatment, the mortality rate remains high. Severe dysfunctions of the disease and the decrease of the body's own ability to remove infection, which is an important cause of the difficulty of controlling infection and multiple organ dysfunction. Neutrophils, as the first line of defense against pathogens, rely on chemotaxis to kill bacteria. In sepsis, under the stimulation of a variety of bacterial toxins and cytokines, neutrality is neutral. With the dysfunction of granulocyte, its recruitment and chemotaxis function is seriously damaged, the phagocytosis ability is decreased, the release of inflammatory mediators increases, and the formation of NETs is increased. It is an important cause of immune dysfunction in patients with sepsis. Therefore, how to regulate the immune function of patients with sepsis and thus effectively play the anti infection effect may be the treatment of sepsis. One of the important means. Lipopolysaccharide (LPS) is a highly acylated sugar fat, located in the outer layer of the Gram-negative (G-) bacilli outer membrane. It is the most important pathogenic ingredient in the endotoxemia and sepsis caused by G- bacillus infection. Early in body and in vitro studies showed that LPS could inhibit the chemotaxis of neutrophils. Adenosine (ADO) is a gland. One of the hydrolysates of purine nucleoside three phosphoric acid (ATP) is a short half-life, but has a powerful effect function.ADO exerts its function through 4 G protein coupled receptors: A1, A2A, A2B and A3 receptors. These 4 ADO receptors are expressed on the surface of neutrophils, and ADO can mediate a variety of functions of neutrophils through its different receptors, including regulation of neutrality. The chemotactic function of granulocytes, while the neutrophil chemotaxis of ADO mediated LPS inhibition is not yet reported. Methods 1. healthy adult volunteers were collected from the elbow vein blood and isolated from healthy adult peripheral blood neutrophils by Ficoll density gradient centrifugation. The Swiss staining, trypan blue staining and flow cytometry were used to detect CD66. B, identify the activity and purity of neutrophils, establish.2. chemotactic model under agarose, use chemotaxis IL-8 and f MLP to detect the directional migration distance of neutrophils, identify chemotaxis and establish chemotaxis using.3. for LPS stimulation of neutrophils, ADO intervention, ADO receptor related inhibitors and agonists, C AMP analogues and Interventions such as inhibitors, AC agonists, p38 MAPK inhibitors, and the use of agarose chemotactic model to detect the directional migration ability of neutrophils under different interventions.4. use flow cytometry and immune imprint (WB) to detect the expression of ADO receptor A1 in neutrophils using protein chip to detect the phosphorylation level of MAPK signal pathway related proteins by protein chip; WB examination. The relative phosphorylation level of the total p38 MAPK was measured. Results the cells separated by 1.Ficoll density gradient centrifugation, the Swiss staining showed that the cell nucleus staining was purple, showing the horseshoe shape or lobulate, the cytoplasm presented light red, neutrophils, trypan blue staining and flow cytometry detection of CD66b, respectively, showing that the activity and purity of the cells were equal to 97%.2., respectively. The chemotactic experiment under agarose showed that the optimal concentration of neutrophil chemotaxis induced by IL-8 and f MLP was 1 mu mol/L and 0.1 micron mol/L.3.LPS stimulated neutrophils, which could obviously inhibit the directed migration of neutrophils to IL-8 or F MLP in a dose-dependent manner, and ADO intervention could restore the chemotaxis of neutrophils inhibited by LPS. Neutrophil chemotaxis mediated by.4.ADO mediated LPS inhibition is mainly mediated by the role of ADO receptor A1 on neutrophil on the neutrophil to stimulate neutrophils, and the expression level of A1 receptor (A1R) is not significantly altered by the A1 receptor (A1R) expression, and the neutrophil chemotactic action of restoring LPS inhibition may not depend on its classic AC-c AMP-P. KA signaling pathway, but inhibit the level of p38 MAPK phosphorylation.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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