可溶性凝集素样受体2在冠状动脉粥样硬化性心脏病中的诊断作用及变化机制的研究

发布时间：2018-05-04 08:40

本文选题：可溶性CLEC-2 + 冠心病　；参考：《苏州大学》2016年博士论文

【摘要】：心血管病是世界范围内导致死亡的主要原因。明确心血管疾病及相关过程的作用机制对于心血管病的及时诊断和治疗具有重要意义,因此在过去的几十年中进行了许多相关的基础和临床的研究。血小板在许多心血管疾病(尤其是急性冠脉综合征)的病理生理机制中扮演着重要的作用,这是由于它们被认为参与到动脉粥样斑块破裂后的血栓形成中。凝集素样受体2 (CLEC-2)是位于人12号染色体C型凝集素受体家族成员,高表达于巨核细胞和血小板中,被认为参与包括血小板活化,淋巴管血管的分化发育等多种生物学过程中。我们之前的研究证实小鼠CLEC-2存在着不同长度的剪切体。此外,全长的CLEC-2能够水解形成可溶性的形式。因此,可溶性形式的受体能够与配体相互作用,抑制配体与细胞表面受体的结合,从而调控病理生理的过程。然而,目前有关CLEC-2在冠状动脉粥样硬化性心脏病中的作用并不明确,这就成为了进行本项研究的初衷。第一部分血浆sCLEC-2对冠状动脉粥样硬化性心脏病的诊断作用目的：明确血浆sCLEC-2在冠状动脉粥样硬化性心脏病中的水平变化及其在冠心病中的临床意义。方法：连续选取2011年5月至2016年10月间在苏州大学附属第一医院、中国医科大附属第一医院心内科住院的冠心病患者146例,其中非急性冠脉综合征患者(Non-ACS)14例,急性冠脉综合征(ACS)132例。正常对照组31例。抽取患者及对照个体的静脉血,采用竞争抑制酶联免疫吸附法(ELISA)测定血浆sCLEC-2浓度及sGPVI浓度。比较血浆sCLEC-2在冠心病患者及健康对照个体中的水平差异,并通过亚组分析,明确sCLEC-2在Non-ACS、ACS及对照组中的水平差异：同时比较不同冠脉病变支数患者间血浆sCLEC-2的差异。GRACE评分对ACS患者进行危险分级后比较不同风险组患者间血浆sCLEC-2的差异。进一步通过二元Logistic回归方法,分析血浆sCLEC-2升高是否为ACS的独立危险因素。采用受试者操作曲线(ROC)曲线分析,评价血浆sCLEC-2对冠心病的诊断价值。通过Pearson线性相关方法,评价血浆sCLEC-2和经典血小板活化指标-血浆sGPVI的关联。结果：冠心病组患者的血浆sCLEC-2水平显著高于对照组(181.86±146.21 pg/mL vs.121.68±56.24 pg/mL, P0.001)。亚组分析显示,对照组、Non-ACS组、ACS患者的血浆sCLEC-2浓度分别为121.68±56.24 pg/mL,126.47±60.98 pg/mL、 187.74±151.42 pg/mL, ACS组血浆sCLEC-2水平较Non-ACS组患者(P0.05)和对照组明显升高(P0.001),而Non-ACS组患者血浆sCLEC-2水平较对照组无明显升高(P0.05)。在冠心病患者中冠状动脉单支病变、双支病变、三支病变患者三组的血浆sCLEC-2浓度分别是196.98±161.44 pg/mL、151.73±118.79 pg/mL、 190.29±145.87pg/mL,三组间血浆sCLEC-2水平无统计学差异(P0.05)。根据GRACE评分,将患者分为低危组、中危组、高危组,三组患者血浆sCLEC-2浓度依次为205.46±185.98pg/mL、177.83±137.53 pg/mL、190.37±149.48 pg/mL,三组间血浆sCLEC-2水平无统计学差异(P0.05)。二元logistic回归分析显示,高血浆sCLEC-2 (OR= 1.006,95% CI:1.000～1.012, P= 0.035).受试者操作曲线(ROC)分析显示,高血浆sCLEC-2水平对冠心病具有一定的诊断效能,ROC曲线下面积(AUC)为0.627,95% CI：0.525～0.728,界点193.65 pg/mL,敏感度为25.3%,特异度为96.8%(P0.05)。Pearson线性相关分析显示,血浆sCLEC-2水平与血小板活化指标-血浆sGPVI i农度呈显著正相关(r=0.474,P0.001)。结论：冠心病患者血浆sCLEC-2水平显著升高,其中ACS患者sCLEC-2水平显著高于Non-ACS患者和健康人群。高血浆sCLEC-2水平是ACS发病的独立危险因素。高血浆sCLEC-2浓度可作为冠心病的有效诊断指标。血浆sCLEC-2与经典血小板表面活化指标GPVI具有良好相关性,可能可以作为血小板活化的新监测指标。第二部分血浆可溶性CLEC-2水平变化机制研究目的：明确血浆可溶性CLEC-2水平变化的可能的原因及相关的信号通路机制。方法：流式细胞术检测血小板及多种免疫细胞CLEC-2的表达情况。分离健康人洗涤血小板,体外给予50 μg/mL氧化低密度脂蛋白(ox-LDL)刺激后,流式细胞术检测平均荧光强度明确CLEC-2从血小板表面脱落；同时利用激酶抑制剂PP2、PRT-060318及Ro-31-8820联合ox-LDL刺激血小板明确影响CLEC-2从血小板表面脱落的信号通路。采用凝血酶联合ox-LDL及8-pCPT-cGMP刺激洗涤血小板,ELISA分析上清中CLEC-2表达水平的变化；在阻断分泌放大信号及血小板整合素外向内信号的条件下,采用MnTMPyP或PP2或Ro-31-8220处理,明确参与oxLDL调控血小板分泌CLEC-2的信号通路。结果：CLEC-2仅在血小板表面出现高表达,而在包括树突状细胞、B细胞、T细胞、单核细胞及中性粒细胞等免疫细胞表面低表达。ox-LDL刺激后血小板表面CLEC-2的脱落,这种效应是通过Src家族激酶-Syk-PKC信号通路来得以实现的。PKG-cGMP信号参与到血小板分泌CLEC-2,而该通路上游的ROS、Src家族激酶及PKC也被发现参与到血小板向胞外分泌CLEC-2。结论：血浆可溶性CLEC-2的水平升高可能是通过两条途径得以实现的,分别是通过血小板表面CLEC-2的脱落及血小板颗粒内容物中CLEC-2分泌。这两条通路分别通过Src家族激酶-Syk-PKC信号通路及Src家族激酶-PKC-ROS-cGMP信号得以实现。
[Abstract]:Cardiovascular disease is the leading cause of death worldwide. It is important for the timely diagnosis and treatment of cardiovascular disease to identify the mechanisms of cardiovascular disease and related processes. Many related basic and clinical studies have been conducted over the past decades. The pathophysiological mechanism of coronary syndrome plays an important role in the formation of thrombus after the rupture of atherosclerotic plaque. Lectin like receptor 2 (CLEC-2) is a member of the C type lectin receptor family on the human chromosome, highly expressed in megakaryocytes and platelets, and is believed to be involved in the blood. In many biological processes, such as platelets activation, and differentiation and development of lymphatic vessels. Our previous study confirmed that the mouse CLEC-2 exists a different length of shear body. In addition, the full length of CLEC-2 can hydrolyze to form a soluble form. Therefore, the soluble form receptor can interact with the ligand and inhibit the ligand and the cell surface receptor. However, the current role of CLEC-2 in coronary atherosclerotic heart disease is not clear, and this is the purpose of this study. Part I, the purpose of plasma sCLEC-2 for the diagnosis of coronary atherosclerotic heart disease: the determination of plasma sCLEC-2 in coronary atherosclerosis Changes in the level of sclerosing heart disease and its clinical significance in coronary heart disease. Methods: 146 patients with coronary heart disease hospitalized in First Hospital Affiliated to Suzhou University from May 2011 to October 2016 were selected in the Department of Cardiology, the first hospital of the first hospital of China Medical University, including 14 cases of non acute coronary syndrome (Non-ACS), acute coronary syndrome. 132 cases (ACS) and 31 cases of normal control group. The venous blood of the patients and the control individuals was extracted and the plasma sCLEC-2 concentration and sGPVI concentration were measured by competitive inhibition enzyme linked immunosorbent assay (ELISA). The levels of plasma sCLEC-2 in patients with coronary heart disease and healthy controls were compared, and the sCLEC-2 was determined in Non-ACS, ACS, and pairs by subgroup analysis. The difference in the level in the group: the difference of plasma sCLEC-2 between the patients with different coronary artery disease and the difference of plasma sCLEC-2 in different risk groups after the risk classification of ACS patients. Further, the two yuan Logistic regression method was used to analyze the independent risk factors of whether the plasma sCLEC-2 elevation was ACS. The patient's operation curve (ROC) curve analysis was used to evaluate the diagnostic value of plasma sCLEC-2 for coronary heart disease. The correlation between plasma sCLEC-2 and the classical platelet activation index plasma sGPVI was evaluated by Pearson linear correlation. Results: the plasma sCLEC-2 level in patients with coronary heart disease was significantly higher than that in the control group (181.86 + 146.21 pg/mL vs.121.68 + 56.24 P) G/mL, P0.001). The subgroup analysis showed that the plasma sCLEC-2 concentration in the control group, Non-ACS group and ACS was 121.68 + 56.24 pg/mL, 126.47 + 60.98 pg/mL, 187.74 + 151.42 pg/mL, and the plasma sCLEC-2 level in ACS group was significantly higher than that in the Non-ACS group (P0.05) and the control group. Xian Shenggao (P0.05). In the patients with coronary artery disease, the plasma sCLEC-2 concentration was 196.98 + 161.44 pg/mL, 151.73 + 118.79 pg/mL and 190.29 + 145.87pg/mL in the patients with single coronary artery disease, double branch lesion and three vessel disease. There was no statistical difference between the three groups (P0.05). According to GRACE score, the patients were divided into low risk group and middle risk. The plasma sCLEC-2 concentration in the three groups was 205.46 + 185.98pg/mL, 177.83 + 137.53 pg/mL and 190.37 + 149.48 pg/mL in the high risk group. There was no statistical difference between the three groups (P0.05). Two yuan logistic regression analysis showed that the high plasma sCLEC-2 (OR= 1.006,95% CI:1.000 ~ 1.012, 0.035). The high plasma sCLEC-2 level has a certain diagnostic efficiency for coronary heart disease. The area under the ROC curve (AUC) is 0.627,95% CI:0.525 to 0.728, the boundary point is 193.65 pg/mL, the sensitivity is 25.3%, and the specificity is 96.8% (P0.05).Pearson linear correlation analysis showed that the plasma sCLEC-2 level is significantly positively correlated with the platelet activation index - sGPVI I agro degree (r=). 0.474, P0.001) conclusion: the level of plasma sCLEC-2 in patients with coronary heart disease is significantly higher, of which the level of sCLEC-2 in ACS patients is significantly higher than that of Non-ACS patients and healthy people. High plasma sCLEC-2 level is an independent risk factor for the pathogenesis of ACS. High plasma sCLEC-2 concentration can be used as an effective diagnostic indicator for coronary heart disease. GPVI has good correlation and may be a new monitoring index for platelet activation. Second part of the change mechanism of plasma soluble CLEC-2 level: the possible cause of the change of plasma soluble CLEC-2 level and the related signaling pathway mechanism. Method: flow cytometry detection of platelets and multiple immunization The expression of cell CLEC-2. Separating healthy people from washing platelets and giving 50 mu g/mL oxidized low density lipoprotein (ox-LDL) in vitro, flow cytometry detected the average fluorescence intensity of CLEC-2 from the surface of platelets; meanwhile, PP2, PRT-060318 and Ro-31-8820 combined with ox-LDL stimulated platelets. The signal pathway of platelet surface falling off. Using thrombin combined with ox-LDL and 8-pCPT-cGMP to stimulate the washing of platelets and ELISA to analyze the changes in the expression of CLEC-2 in the supernatant. Under the conditions of blocking the secretory signal and the extroversion of the platelet integrin, the MnTMPyP or PP2 or Ro-31-8220 treatments are used to explicitly participate in the regulation of the platelets in oxLDL. Results: the signal pathway of CLEC-2 is secreted. Results: high expression of CLEC-2 only on the surface of platelets, and the drop of CLEC-2 on the surface of platelets on the surface of immune cells, including dendritic cells, B cells, T cells, monocytes and neutrophils, is expressed by the Src family kinase -Syk-PKC signaling pathway. The present.PKG-cGMP signal is involved in the platelet secretion of CLEC-2, and the ROS, Src family kinase and PKC upstream of the pathway are also found to be involved in the exocrine CLEC-2. conclusion: the level of soluble CLEC-2 in plasma may be achieved through two pathways, respectively, through the abscission of the platelet surface CLEC-2 and the platelets. CLEC-2 is secreted in granule contents. These two pathways are realized through Src family kinase -Syk-PKC signaling pathway and Src family kinase -PKC-ROS-cGMP signal.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.4

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