本文選題：可溶性CLEC-2 + 冠心病��； 參考：《蘇州大學》2016年博士論文

【摘要】：心血管病是世界范圍內導致死亡的主要原因。明確心血管疾病及相關過程的作用機制對于心血管病的及時診斷和治療具有重要意義,因此在過去的幾十年中進行了許多相關的基礎和臨床的研究。血小板在許多心血管疾病(尤其是急性冠脈綜合征)的病理生理機制中扮演著重要的作用,這是由于它們被認為參與到動脈粥樣斑塊破裂后的血栓形成中。凝集素樣受體2 (CLEC-2)是位于人12號染色體C型凝集素受體家族成員,高表達于巨核細胞和血小板中,被認為參與包括血小板活化,淋巴管血管的分化發(fā)育等多種生物學過程中。我們之前的研究證實小鼠CLEC-2存在著不同長度的剪切體。此外,全長的CLEC-2能夠水解形成可溶性的形式。因此,可溶性形式的受體能夠與配體相互作用,抑制配體與細胞表面受體的結合,從而調控病理生理的過程。然而,目前有關CLEC-2在冠狀動脈粥樣硬化性心臟病中的作用并不明確,這就成為了進行本項研究的初衷。第一部分血漿sCLEC-2對冠狀動脈粥樣硬化性心臟病的診斷作用目的：明確血漿sCLEC-2在冠狀動脈粥樣硬化性心臟病中的水平變化及其在冠心病中的臨床意義。方法：連續(xù)選取2011年5月至2016年10月間在蘇州大學附屬第一醫(yī)院、中國醫(yī)科大附屬第一醫(yī)院心內科住院的冠心病患者146例,其中非急性冠脈綜合征患者(Non-ACS)14例,急性冠脈綜合征(ACS)132例。正常對照組31例。抽取患者及對照個體的靜脈血,采用競爭抑制酶聯免疫吸附法(ELISA)測定血漿sCLEC-2濃度及sGPVI濃度。比較血漿sCLEC-2在冠心病患者及健康對照個體中的水平差異,并通過亞組分析,明確sCLEC-2在Non-ACS、ACS及對照組中的水平差異：同時比較不同冠脈病變支數患者間血漿sCLEC-2的差異。GRACE評分對ACS患者進行危險分級后比較不同風險組患者間血漿sCLEC-2的差異。進一步通過二元Logistic回歸方法,分析血漿sCLEC-2升高是否為ACS的獨立危險因素。采用受試者操作曲線(ROC)曲線分析,評價血漿sCLEC-2對冠心病的診斷價值。通過Pearson線性相關方法,評價血漿sCLEC-2和經典血小板活化指標-血漿sGPVI的關聯。結果：冠心病組患者的血漿sCLEC-2水平顯著高于對照組(181.86±146.21 pg/mL vs.121.68±56.24 pg/mL, P0.001)。亞組分析顯示,對照組、Non-ACS組、ACS患者的血漿sCLEC-2濃度分別為121.68±56.24 pg/mL,126.47±60.98 pg/mL、 187.74±151.42 pg/mL, ACS組血漿sCLEC-2水平較Non-ACS組患者(P0.05)和對照組明顯升高(P0.001),而Non-ACS組患者血漿sCLEC-2水平較對照組無明顯升高(P0.05)。在冠心病患者中冠狀動脈單支病變、雙支病變、三支病變患者三組的血漿sCLEC-2濃度分別是196.98±161.44 pg/mL、151.73±118.79 pg/mL、 190.29±145.87pg/mL,三組間血漿sCLEC-2水平無統(tǒng)計學差異(P0.05)。根據GRACE評分,將患者分為低危組、中危組、高危組,三組患者血漿sCLEC-2濃度依次為205.46±185.98pg/mL、177.83±137.53 pg/mL、190.37±149.48 pg/mL,三組間血漿sCLEC-2水平無統(tǒng)計學差異(P0.05)。二元logistic回歸分析顯示,高血漿sCLEC-2 (OR= 1.006,95% CI:1.000～1.012, P= 0.035).受試者操作曲線(ROC)分析顯示,高血漿sCLEC-2水平對冠心病具有一定的診斷效能,ROC曲線下面積(AUC)為0.627,95% CI：0.525～0.728,界點193.65 pg/mL,敏感度為25.3%,特異度為96.8%(P0.05)。Pearson線性相關分析顯示,血漿sCLEC-2水平與血小板活化指標-血漿sGPVI i農度呈顯著正相關(r=0.474,P0.001)。結論：冠心病患者血漿sCLEC-2水平顯著升高,其中ACS患者sCLEC-2水平顯著高于Non-ACS患者和健康人群。高血漿sCLEC-2水平是ACS發(fā)病的獨立危險因素。高血漿sCLEC-2濃度可作為冠心病的有效診斷指標。血漿sCLEC-2與經典血小板表面活化指標GPVI具有良好相關性,可能可以作為血小板活化的新監(jiān)測指標。第二部分血漿可溶性CLEC-2水平變化機制研究目的：明確血漿可溶性CLEC-2水平變化的可能的原因及相關的信號通路機制。方法：流式細胞術檢測血小板及多種免疫細胞CLEC-2的表達情況。分離健康人洗滌血小板,體外給予50 μg/mL氧化低密度脂蛋白(ox-LDL)刺激后,流式細胞術檢測平均熒光強度明確CLEC-2從血小板表面脫落；同時利用激酶抑制劑PP2、PRT-060318及Ro-31-8820聯合ox-LDL刺激血小板明確影響CLEC-2從血小板表面脫落的信號通路。采用凝血酶聯合ox-LDL及8-pCPT-cGMP刺激洗滌血小板,ELISA分析上清中CLEC-2表達水平的變化；在阻斷分泌放大信號及血小板整合素外向內信號的條件下,采用MnTMPyP或PP2或Ro-31-8220處理,明確參與oxLDL調控血小板分泌CLEC-2的信號通路。結果：CLEC-2僅在血小板表面出現高表達,而在包括樹突狀細胞、B細胞、T細胞、單核細胞及中性粒細胞等免疫細胞表面低表達。ox-LDL刺激后血小板表面CLEC-2的脫落,這種效應是通過Src家族激酶-Syk-PKC信號通路來得以實現的。PKG-cGMP信號參與到血小板分泌CLEC-2,而該通路上游的ROS、Src家族激酶及PKC也被發(fā)現參與到血小板向胞外分泌CLEC-2。結論：血漿可溶性CLEC-2的水平升高可能是通過兩條途徑得以實現的,分別是通過血小板表面CLEC-2的脫落及血小板顆粒內容物中CLEC-2分泌。這兩條通路分別通過Src家族激酶-Syk-PKC信號通路及Src家族激酶-PKC-ROS-cGMP信號得以實現。
[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.

【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R541.4

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1 Gergely Feher;Andrea Feher;Gabriella Pusch;Katalin Koltai;Antal Tibold;Beata Gasztonyi;Elod Papp;Laszlo Szapary;Gabor Kesmarky;Kalman Toth;;Clinical importance of aspirin and clopidogrel resistance[J];World Journal of Cardiology;2010年07期

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