冠心病患者尿激酶型纤溶酶原激活物受体（uPAR）与黏附分子CD11b/CD18表达水平及对单核细胞黏附功能影响研究

【作者】 杨倩；

【导师】 戴玉华； 方全；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 背景:纤溶系统不仅促使纤维蛋白的溶解,而且参与了很多病理、生理过程,例如细胞迁移、组织重塑。纤溶系统在炎症、新生血管形成、动脉粥样硬化过程中也起到重要作用。尿激酶型纤溶酶原激活物受体(uPAR)是纤溶系统中较为重要的成分。uPAR可以与细胞胞外及细胞膜上一些分子结合,例如尿激酶型纤溶酶原激活物(uPA)、玻璃体粘连蛋白(VN)、整合素等。uPAR除了可以激活细胞周围蛋白水解还参与细胞黏附、细胞增殖和趋化等过程,这些生物学功能贯穿于动脉粥样硬化发生演变的过程中。越来越多证据表明,局部和全身炎症在动脉粥样硬化发展中起着重要作用。白细胞黏附到血管内皮细胞是动脉粥样硬化最早期的病理变化,白细胞跨内皮迁移至血管壁间隙是组织损伤和炎症反应的必要步骤,白细胞与血管内皮的黏附是此过程的中最重要的一环。CD11b/CD18是整合素家族中一员,它作为uPAR配体可与uPAR形成复合体结构介导细胞黏附过程。白细胞与内皮细胞之间的黏附是通过CD11b/CD18与其另一配体—内皮细胞表达的细胞间粘附分子-1(ICAM-1,CD54)相互作用而介导完成的。烟酰胺腺嘌呤二核苷酸糖原水解酶(NADG)、磷脂酰肌醇磷脂酶C(PI-PLC)可以破坏uPAR与CD11b/CD18形成的复合体结构。目的:研究不同临床类型冠心病患者外周血单核细胞和中性粒细胞上uPAR、CD11b/CD18的表达水平及单核细胞黏附功能,探讨氧化型低密度脂蛋白(oxLDL)和高浓度葡萄糖对单核uPAR、CD11b/CD18的表达和黏附功能的影响并比较抗uPAR抗体、抗CD11b抗体、NADG、PI-PLC、抗ICAM-1抗体对单核细胞黏附功能的抑制作用。方法:研究对象包括在协和医院门急诊及住院的急性心梗患者26例,陈旧心梗患者患者23例,健康志愿者20例,用流式细胞仪测量外周血单核细胞和中性粒细胞上uPAR、CD11b/CD18表达的荧光强度和比例。采用密度梯度离心法和粘附法分离纯化冠心不颊咄庵苎ズ讼赴?加入oxLDL和高浓度葡萄糖刺激体外培养的单核细胞,用ELISA法检测单核细胞产生的uPAR及CD11b/CD18抗原。并将第二部分用oxLDL和高浓度葡萄糖体外刺激培养的单核细胞与脐静脉内皮细胞黏附,比较抗uPAR抗体、抗CD11b抗体、NADG、PI-PLC、抗ICAM-1抗体等对黏附功能的抑制作用。结果:急性心梗患者外周血单核、中性粒细胞上uPAR、CD11b/CD18的表达水平均较陈旧心梗患者及正常人组显著性升高(p＜0.05)。陈旧心梗患者与正常人外周血单核、中性粒细胞上uPAR、CD11b/CD18的表达水平之间没有显著性差异。在体外实验中,oxLDL与高浓度的葡萄糖可以显著上调正常人及陈旧心梗患者单核细胞上uPAR、CD11b/CD18的表达(p＜0.05),并增强单核细胞对内皮细胞黏附能力。急性.心梗患者外周血单核细胞对内皮细胞黏附能力均较陈旧心梗患者及正常人组显著性增强(p＜0.05)。陈旧心梗患者与正常人外周血单核细胞对内皮细胞黏附能力之间没有显著性差异。抗uPAR抗体、抗CD11b抗体的应用可有效降低单核细胞对内皮细胞的黏附能力。NADG、PI-PLC、抗ICAM-1抗体对单核细胞的黏附功能明显抑制,各组不同患者、不同刺激物培养的单核细胞黏附率下降到几乎相近的水平。结论:本研究证实急性心梗患者单核细胞、中性粒细胞上uPAR、CD11b/CD18表达水平较陈旧心梗患者及正常人显著升高,单核细胞黏附功能显著增强。陈旧心梗患者与正常人在单核细胞uPAR、CD11b/CD18表达及单核细胞黏附功能上均没有显著差别。高糖、高脂可刺激体外单核细胞uPAR、CD11b/CD18表达及使单核细胞黏附功能显著增强。本研究通过体外实验探讨了冠心病两大危险因素高糖、高脂对单核细胞uPAR、CD11b/CD18表达及黏附功能重要影响作用,并通过不同刺激干扰有效降低单核细胞黏附功能,这些结果为进一步深入研究uPAR、CD11b/CD18表达在动脉粥样硬化形成中的机制打下了基础,并可能为临床动脉粥样硬化预防、治疗带来新的治疗方向和靶点。更多还原

【Abstract】 Background:The urokinase-mediated plasminogen activaton system is involved in many physiologic and pathological events that include cell migration and tissue remodeling. This system also plays an important role in the process such as inflammation, angiogenesis, atherogenesis and tumor invation and metastasis. The urokinase-type plasminogen activator receptor(uPAR) is a key molecular of this system and can bind extracellular and cell membrane molecular such as urokinase type plasminogen activator(uPA), vitronectin(VN), integrins and so on. Independently of cell suface-associated proteolysis, uPAR is involved in several cellular functions like adhesion, proliferation and chemotaxis, all of which are fundamental processes in atherogensis. More and more studies show that local and systemic inflammation plays an important role in the development of atherogensis. Leukocytes adhesion to vascular endothelial cells is the earliest pathologicalchange in atherosclerosis and Leukocyte migration through endothelial cells to the vessel wall space is the necessary stept to issue damage and inflammation. During this process, the leukocytes adhesion to vascular endothelial is the most important part. CD11b/CD18, which belongs to the family of integrins, is a kind of uPAR’s ligand. It can form complexs with uPAR to mediate cell adhesion. Leukocytes adhesion on endothelial cells is based on the interaction between CD lib/CD 18 and its ligand - intercellular adhesion molecule-1 (ICAM-1, CD54) which is expressed on endothelial cells. Phosphatidylinositolspecific phosphlipase C (PI-PLC) and nicotinamide adenine dinucleotide glycohydrolase (NADG) can break the complexs from the cell surface.Objective:Our study was aiming to evaluate the atherogenic role of uPAR, CD11b/CD18 expressed on human monocytes and neutrophilic granulocytes, and the influence on the capacity of monocyte adhesion. We tried to investigate the effects of oxLDL and high glucose on uPAR, CD11b/CD18 expression on monocyte, and also tried to investigate the different influences on monocyte binding capacity for human umbilical vein endothelial cells between uPAR antibody, CD11b antibody, NADG, PI-PLC and ICAM-1 antibodyMethods:The study group comprised 26 patients presenting with AMI within 24 hours after the onset of pain. 23 patients with OMI, and 20 healthy volunteers were studied as control group. Mean fluorescence intensity and the proportion of peripheral monocytes and neutrophilic granulocytes expressing uPAR, CD11b/CD18 were measured by flow cytometer. Monocytes were separated and purified by density gradient centrifugation and adhering assay. In vitro, we incubated the cells with oxLDL and high glucose, and we measured uPAR, CD11b/CD18 by ELISA. Anti-uPAR, anti-CD11b, NADG, PI-PLC, anti-ICAM-1 were incubated with monocytes together to investigate their different influences on monocyte binding capacity for human umbilical vein endothelial cells.Results:Surface expression of uPAR and CD11b/CD18 were both significantly higher on peripheral monocytes and neutrophilic granulocytes in patients with AMI, compared with that in patients with OMI and healthy volunteers (p<0.05). Neither surface expression of uPAR nor CD11b/CD18 had significant differences between the patients with OMI and healthy volunteers. In vitro, oxLDL and high glucose could significantly increased the expression of uPAR and CD11b/CD18 on monocytes, and also enhanced the capacity of monocyte adhesiveness compared with that of control group (p<0.05). The capacity of monocytes binding for human umbilical vein endothelial cells was significantly stronger in patients with AMI compared with that in patients with OMI and healthy volunteers (p<0.05). There are no significant differences between the patients with OMI and healthy volunteers on the capacity of monocytes binding for human umbilical vein endothelial cells. Adhesion of monocytes was effectively inhibited by anti-uPAR and anti-CD11b. Adhesion of monocytes was almost inhibited to a same level especially by NADG, PI-PLC, anti-ICAM-1.Conclusions: Our study showed both uPAR and CD11b/CD18 surface expression on monocytes and neutrophilic granulocytes in patients with AMI were significantly increased compared with that in the patients with OMI and healthy volunteers. At the same time, the capacity of monocytes adhesion was also significantly stronger in patients with AMI. There are no significant differences between the patients with OMI and healthy volunteers either in uPAR, CD11b/CD18 expression or in capacity of monocytes adhesion. OxLDL and high glucose not only increased the expression of uPAR and CD11b/CD 18 on monocytes significantly, but also enhanced the capacity of monocyte adhesion. Our study showed hyperlipidemia and pathoglycemia, which are two important high risk to coronary artery disease, were probably a critical pair of factors in activating uPAR,CD11b/CD18 expression and enhancing capacity of adhesion on monocytes. Adhesion of monocyte could be effectively inhibited by using several antibodys. Our results provided new knowledge of monocytes atherogenic role by uPAR and CD11b/CD18, and further investigation was required to elucidate its mechanism and the possibility of clinical application. These might provide a new way for preventing and treating coronary artery disease.更多还原