AF-1对于内毒素诱导的中性粒细胞与内皮细胞粘附的影响及机制研究

发布时间：2018-02-15 00:40

本文关键词： 中性粒细胞 Percoll密度梯度离心法 Ficoll-Hypaque密度梯度离心法裂解红细胞法 Dextran红细胞沉降法 Antiflammin-1(AF-1) 粘附中性粒细胞内皮细胞　出处：《中南大学》2008年硕士论文　论文类型：学位论文

【摘要】： 第一章人外周血中性粒细胞不同分离方法的比较目的:从血液中分离中性粒细胞(neutrophil)是在细胞水平研究其生物学特性及功能的基础。根据目前国内外文献资料显示的常用中性粒细胞分离方法,选取四种,即Percoll非连续密度梯度离心法、Ficoll-Hypaque密度梯度离心法、裂解红细胞法、Dextran作用下红细胞自然沉降法进行细胞纯度、回收率、存活率比较,旨在寻找一种简单、高效的中性粒细胞分离方法。方法:采取健康人外周静脉血,分别采用以上四种方法进行中性粒细胞分离,对分离纯化的中性粒细胞纯度、回收率、存活率进行比较。结果:Percoll非连续密度梯度离心法与Ficoll-Hypaque密度梯度离心法分离得到的细胞纯度均大于90%,两者间比较无显著差异(P＞0.05);裂解红细胞法和Dextran作用下红细胞自然沉降法分离得到的细胞纯度略低于Percoll非连续密度梯度离心法和Ficoll-Hypaque密度梯度离心法(P＜0.05或P＜0.01);Percoll非连续密度梯度离心法、Ficoll-Hypaque密度梯度离心法和裂解红细胞法的回收率均高于Dextran作用下红细胞自然沉降法(P＜0.01或P＜0.05);Percoll非连续密度梯度离心法回收的中性粒细胞存活率明显高于其它三组(P＜0.01或P＜0.05)。结论:Percoll非连续密度梯度离心法分离中性粒细胞,纯化程度好,回收率和细胞存活率高,是一种简单、高效的中性粒细胞分离方法,适于临床和科研中广泛应用。第二章AF-1对于内毒素诱导的中性粒细胞与内皮细胞粘附的影响及机制研究目的:中性粒细胞(neutrophil)在肺内聚集是急性肺损伤(acutelung injury,ALI)发病的最初环节,中性粒细胞与肺血管内皮细胞粘附是炎症反应的最初现象,是其进一步移行入肺组织的基础。子宫珠蛋白(uteroglobin,UG)是一种多功能非类固醇类激素蛋白,具有抗炎、抗氧化、免疫调节、抑制肿瘤发生等作用。来源于子宫珠蛋白C末端第三个α螺旋的九肽Antiflammin-1(AF-1),也被证实具有强烈的抗炎作用。关于AF-1抗炎作用的机制至今尚未完全明了,因此本研究旨在通过观察AF-1对于内毒素诱导的中性粒细胞与内皮细胞粘附及粘附分子表达的影响,检测中性粒细胞和内皮细胞子宫珠蛋白结合蛋白(Uteroglobin-binding protein,UGBP)的表达情况,通过应用制备的抗-UGBP抗体观察抗-UGBP抗体对AF-1粘附抑制及粘附分子表达的影响,还通过检测AF-1、抗-UGBP抗体对于P38/MAPK信号转导通路的影响,初步探讨AF-1产生抗炎作用的机制,为ALI的治疗提供新的靶点。方法:采取健康人外周静脉血分离纯化中性粒细胞,培养人脐静脉内皮细胞(HUVEC-12),分别应用不同浓度、不同作用时间的AF-1预处理中性粒细胞和内皮细胞,观察中性粒细胞与内皮细胞粘附率的变化及应用流式细胞仪检测粘附分子表达的变化;通过应用免疫荧光、流式细胞术、RT-PCR一系列实验技术检测中性粒细胞和内皮细胞UGBP的表达情况;应用制备的抗-UGBP抗体预处理观察抗-UGBP多抗对AF-1粘附抑制及粘附分子表达的影响;进一步应用Western-Blot技术检测AF-1、抗-UGBP抗体对于P38/MAPK信号转导通路的影响。结果: 1.应用AF-1(100μmol/L)分别预处理内皮细胞、中性粒细胞15min～2h时可以明显抑制内毒素诱导的中性粒细胞与内皮细胞的粘附,与LPS组比较均有统计学意义(P＜0.01),其中以30min预处理抑制作用最明显,随着预处理时间延长,其粘附抑制效应均降低,预处理时间为4h时,其粘附抑制作用与LPS组比较没有统计学差异;不同浓度AF-1分别预处理内皮细胞、中性粒细胞30min,研究发现AF-1 1μmol/L对中性粒细胞与内皮细胞的粘附即产生抑制作用(P＜0.05),随着AF-1浓度增高其粘附抑制作用逐渐增强,呈剂量依赖性。流式细胞仪检测结果发现AF-1可以抑制内毒素诱导的中性粒细胞粘附分子CD11b和内皮细胞粘附分子CD54的表达,而AF-1本身对于静息状态下的中性粒细胞的CD11b和内皮细胞的CD54表达没有影响。 2.免疫荧光、RT-PCR、流式细胞实验均证实在内皮细胞和中性粒细胞上均存在UGBP的表达,并且中性粒细胞的表达强于内皮细胞。 3.粘附率测定及粘附分子表达测定结果显示抗-UGBP抗体预处理可以抑制AF-1的粘附抑制效应。 4.进一步应用Western-Blot检测AF-1、抗-UGBP抗体对于P38/MAPK磷酸化水平的影响。结果显示,AF-1可以明显下调内毒素诱导的内皮细胞P38/MAPK的磷酸化水平,抗-UGBP抗体可以抑制AF-1作用引起的下调效应。结论: 1.AF-1可以抑制内毒素诱导的中性粒细胞与内皮细胞的粘附及粘附分子表达,说明AF-1抗炎作用的产生与调节粘附分子的表达有关系。 2.中性粒细胞和内皮细胞均有UGBP表达,以细胞膜表达最为强烈,细胞浆内也有少量表达,并且中性粒细胞UGBP的表达水平高于内皮细胞。 3.抗-UGBP抗体预处理可以抑制AF-1对于粘附的抑制作用及粘附分子表达变化,说明AF-1的粘附抑制作用同UGBP密切相关。 4.AF-1可以通过与UGBP结合调节细胞内P38/MAPK磷酸化水平而影响细胞内信号转导。因此我们认为AF-1通过结合细胞表面UGBP进而调节细胞内信号通路P38/MAPK的磷酸化水平而抑制中性粒细胞与内皮细胞的粘附及粘附分子的表达,产生抗炎作用。
[Abstract]:A comparison of different methods of separation of neutrophils in human peripheral blood
Objective: to isolate neutrophils from the blood (neutrophil) is the base of the research on the biological characteristics and function at the cellular level. According to the separation method commonly used at home and abroad literature shows that the neutral granulocyte, select four kinds, namely Percoll discontinuous density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation, red blood cell lysis method. The effect of Dextran natural erythrocyte sedimentation method cell purity, recovery rate, survival rate, in order to find a simple and efficient method for the separation of neutrophils.
Methods: peripheral blood was taken from healthy people. Neutrophils were separated by the above four methods. The purity, recovery rate and survival rate of purified neutrophils were compared.
Results: the Percoll non isolated cells purity continuous density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation were higher than 90%, there is no significant difference between them (P > 0.05); erythrocyte lysis method and Dextran effect of natural erythrocyte sedimentation method isolated cell purity was slightly lower than Percoll discontinuous density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation (P < 0.05 or P < 0.01); Percoll discontinuous density gradient centrifugation. The recovery of Ficoll-Hypaque density gradient centrifugation and erythrocyte lysis method were higher than the natural erythrocyte sedimentation under the action of Dextran (P < 0.01 or P < 0.05); neutrophil Percoll discontinuous density gradient centrifugal recovery survival rate was significantly higher than the other three groups (P < 0.01 or P < 0.05).
Conclusion: Percoll discontinuous density gradient centrifugation is a simple and effective method for isolation of neutrophils. It is suitable for clinical and scientific research.
The effect and mechanism of second chapter AF-1 on endotoxin induced neutrophils adhesion to endothelial cells
Objective: neutrophil (neutrophil) in the lung of acute lung injury is the aggregation (acutelung injury ALI) the first link of the incidence of neutropenia and pulmonary vascular endothelial cell adhesion is the initial phenomenon of inflammatory reaction, is the basis of further migration into the lung tissue. Uteroglobin (Uteroglobin, UG) is a kind of multifunctional non steroid hormone protein, anti-inflammatory, antioxidant, immune regulation, tumor suppression effect. Nine peptide Antiflammin-1 derived from the Uteroglobin C terminal third alpha helix (AF-1), has also been shown to have anti-inflammatory effects strongly. On the mechanism of the anti-inflammatory effect of AF-1 is not yet fully understood, so this study in order to effect by observing the AF-1 for endotoxin induced neutrophil adhesion to endothelial cells and the expression of adhesion molecules, detection of neutrophils and endothelial cells (Uteroglobin binding protein Uteroglobin-b Inding protein, UGBP) expression, through the application of the preparation of anti -UGBP antibody to observe the effect of anti -UGBP antibody on the expression of AF-1 inhibited the adhesion and adhesion molecules, but also through the detection of AF-1, effects of anti -UGBP antibody for P38/MAPK signal transduction pathway, to explore the mechanism of AF-1 anti inflammatory effect, provide a new target for the treatment of ALI.
Methods: healthy human peripheral venous blood purification of neutrophils, cultured human umbilical vein endothelial cells (HUVEC-12), respectively with different concentrations, different time of AF-1 pretreatment of neutrophils and endothelial cells, to observe the changes of neutrophil adhesion and changes of endothelial cells and the rate of application of flow cytometry to detect the expression of adhesion molecules; by using immunofluorescence and flow cytometry, the expression of RT-PCR in a series of experimental techniques for the detection of neutrophils and endothelial cells UGBP; application of the preparation of anti -UGBP antibody pretreatment effects of anti -UGBP anti adhesion inhibition on the expression of AF-1 and adhesion molecules; the further application of AF-1 Western-Blot detection technology, influence anti -UGBP antibodies to P38/MAPK signal transduction pathway.
Result:
Application of 1. AF-1 (100 mol/L) were treated with endothelial cells, adhesion of neutrophil 15min ~ 2H could inhibit LPS induced neutrophil and endothelial cells, compared with LPS group were statistically significant (P < 0.01), of which 30min pretreatment inhibited the most obvious, with the increasing of pretreatment time the inhibitory effect was decreased, adhesion, pretreatment time was 4h, the inhibition compared with the LPS group had no significant difference; different concentrations of AF-1 were treated with endothelial cells, neutrophils, 30min, the study found that the adhesion of AF-1 1 mol/L on neutrophils and endothelial cells is inhibited (P < 0.05), with inhibition of AF-1 concentration increased the adhesion increased gradually in a dose-dependent manner. Flow cytometry results showed that neutrophil adhesion molecule CD11b and AF-1 can inhibit the adhesion of endothelial cells induced by lipopolysaccharide The expression of molecular CD54, while AF-1 itself has no effect on the CD54 expression of CD11b and endothelial cells in resting state neutrophils.
2. immunofluorescence, RT-PCR and flow cytometry demonstrated that UGBP expression was present on endothelial cells and neutrophils, and the expression of neutrophils was stronger than that of endothelial cells.
3. determination of adhesion rate and expression of adhesion molecules showed that anti -UGBP antibody preconditioning could inhibit the adhesion inhibition effect of AF-1.
4., we further applied Western-Blot to detect AF-1 and the effect of anti -UGBP antibody on P38/MAPK phosphorylation level. The results showed that AF-1 could significantly reduce the level of P38/MAPK induced by endotoxin, and -UGBP antibody could inhibit the down-regulation effect induced by AF-1.
Conclusion:
1.AF-1 can inhibit the adhesion of neutrophils to endothelial cells and expression of adhesion molecules induced by endotoxin, indicating that the production of AF-1 is related to regulating the expression of adhesion molecules.
2. neutrophils and endothelial cells had UGBP expression, the most strongly expressed in cell membrane, and a few in cytoplasm, and the expression level of UGBP in neutrophils was higher than that in endothelial cells.
3. anti -UGBP antibody pretreatment could inhibit the inhibitory effect of AF-1 on adhesion and the changes in the expression of adhesion molecules, indicating that the adhesion inhibition of AF-1 is closely related to UGBP.
4.AF-1 can affect intracellular signal transduction by modulating the level of P38/MAPK phosphorylation in cells with UGBP.
Therefore, we think that AF-1 can regulate the level of phosphorylation of intracellular signaling pathway P38/MAPK and inhibit the expression of adhesion molecules and neutrophils, and produce anti-inflammatory effect by binding the cell surface UGBP to further regulate the level of phosphorylation of intracellular signaling pathway.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【引证文献】