本文選題：膿毒癥 + 中性粒細胞��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：背景與目的膿毒癥是宿主對微生物感染的反應(yīng)失調(diào)導(dǎo)致了危及生命的器官功能障礙,是感染、燒/創(chuàng)傷、休克等急危重患者的嚴重并發(fā)癥。隨著當(dāng)代醫(yī)療技術(shù)、危重病監(jiān)護手段、治療措施的進步,但其病死率依然居高不下。膿毒癥時,宿主免疫功能嚴重失調(diào),機體自身清除感染能力下降,是導(dǎo)致感染難以控制和多器官功能障礙的重要原因。中性粒細胞作為防御病原體的第一道防線,其依賴趨化功能來發(fā)揮殺滅細菌作用。膿毒癥時,在多種細菌毒素及細胞因子的刺激下,中性粒細胞功能失調(diào),其招募和趨化功能嚴重受損,吞噬能力下降,炎癥介質(zhì)釋放增多,NETs形成增加,是膿毒癥患者免疫功能紊亂的重要原因。因此,如何調(diào)控膿毒癥患者自身的免疫功能,從而有效的發(fā)揮抗感染的作用,可能成為治療膿毒癥的一個重要手段之一。脂多糖(LPS)是一種高度�；奶侵�,位于革蘭氏陰性(G-)桿菌外膜的外層,是G-桿菌感染所致的內(nèi)毒素血癥及膿毒癥時最重要的致病成分。早期在體和體外研究均表明LPS能夠抑制中性粒細胞的趨化功能。腺苷(ADO)是腺嘌呤核苷三磷酸(ATP)的水解產(chǎn)物之一,半衰期很短,但具有強大的效應(yīng)功能。ADO發(fā)揮其功能是通過4種G蛋白偶聯(lián)受體:A1、A2A、A2B和A3受體。這4種ADO受體均在中性粒細胞表面表達,ADO可通過其不同的受體介導(dǎo)中性粒細胞多種功能包括調(diào)控中性粒細胞的趨化功能,而關(guān)于ADO介導(dǎo)的恢復(fù)LPS抑制的中性粒細胞趨化作用目前尚無報道。方法1.采集健康成年志愿者肘部靜脈血,通過Ficoll密度梯度離心法分離健康成人外周血中性粒細胞;采用瑞士染色、臺盼藍染色及流式細胞術(shù)檢測CD66b,鑒定中性粒細胞的活性及純度。2.建立瓊脂糖下趨化模型,使用趨化物IL-8和f MLP檢測中性粒細胞的定向遷移距離,鑒定趨化模型及確立趨化物的使用方案。3.對中性粒細胞進行LPS刺激、ADO干預(yù)、ADO受體相關(guān)抑制劑和激動劑、c AMP類似物和抑制劑、AC激動劑、p38 MAPK抑制劑等干預(yù),采用瓊脂糖下趨化模型檢測不同干預(yù)下中性粒細胞的定向遷移能力。4.采用流式細胞術(shù)和免疫印記(WB)檢測中性粒細胞ADO受體A1的表達。5.采用蛋白芯片檢測MAPK信號通路相關(guān)蛋白的磷酸化水平;WB檢測總p38 MAPK相對磷酸化水平。結(jié)果1.Ficoll密度梯度離心法分離的細胞,瑞士染色發(fā)現(xiàn)細胞核染色為紫色,呈現(xiàn)馬蹄形或分葉狀,細胞胞質(zhì)呈現(xiàn)淺紅色,為中性粒細胞;臺盼藍染色及流式細胞術(shù)檢測CD66b,分別顯示細胞的活性和純度均≥97%。2.瓊脂糖下趨化實驗顯示IL-8和f MLP誘導(dǎo)中性粒細胞趨化的最適濃度分別為1μmol/L和0.1μmol/L。3.LPS刺激中性粒細胞后,可以明顯抑制中性粒細胞定向遷移到IL-8或f MLP,呈劑量依賴方式;ADO干預(yù)能夠恢復(fù)LPS抑制的中性粒細胞的趨化,呈濃度依賴方式。4.ADO介導(dǎo)的恢復(fù)LPS抑制的中性粒細胞趨化主要是通過中性粒細胞表面的ADO受體A1發(fā)揮作用。5.LPS刺激中性粒細胞后,其對A1受體(A1R)表達水平未見明顯改變。6.A1R發(fā)揮恢復(fù)LPS抑制的中性粒細胞趨化作用可能不依賴其經(jīng)典的AC-c AMP-PKA信號通路,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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7

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