【作者】 费敏；

【导师】 阮长耿；

【作者基本信息】 苏州大学 ， 内科学（血液病学）， 2016， 博士

【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, P<0.001)。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(P<0.05)和对照组明显升高(P<0.001),而Non-ACS组患者血浆sCLEC-2水平较对照组无明显升高(P>0.05)。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(P>0.05)。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(P>0.05)。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%(P<0.05)。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,P<0.001)。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。更多还原

【Abstract】 Cardiovascular diseases (CVDs) are the leading cause of death all over the world. Understanding of mechanism underlying CVD has become a priority task for early diagnosis and effective treatment. Consequently, the last decades have seen an astonishing amount of studies in this field. Importantly, the obligatory roles of platelets in CVD is hallmarked by their involvement in the thrombus formation after rupture of atherosclerotic plaques. Being a member of C-type lectin receptor family encoded by the gene CLEC1B located on human chromosome 12, CLEC-2 is highly expressed in megakaryocytes and platelets. A growing body of evidence has demonstrated the important roles of CLEC-2 in a variety of physiological processes including platelet activation and lymphatic development. We previously showed two isoforms of mouse CLEC-2 due to differential cleavage. Full length CLEC-2 can be hydrolyzed into a soluble form, which binds to the ligands of CLEC-2 and inhibits membrane-bound CLEC-2 activation. However, whether CLEC-2 plays a role in coronary atherosclerotic heart disease (CHD) remains largely unknown.Part I Diagnostic value of plasma soluble CLEC-2 in CHDObjective:To evaluate the plasma level and of soluble CLEC-2 in CHD patients and explore the clinical significance of CLEC-2 in CHD.Methods:One hundred and forty-six patients with CHD, including 14 cases of non-ACS and 132 cases of ACS, hospitalized in the first affiliated hospital of Soochow University and first affiliated hospital to Chinese Medical University from May 2011 through October 2016 were enrolled in the study. The control group contains 31 healthy subjects. All patients and control subjects underwent venous blood collection to detect plasma sCLEC-2 and sGPVI levels using the enzyme-linked immunosorbent assay (ELISA). Plasma levels of sCLEC-2 were compared between patients with CHD and healthy controls. Subgroup analysis was performed to compare serum sCLEC-2 levels among ACS patients, non-ACS patients and healthy controls. Comparison of sCLEC-2 level was also carried out among ACS patients with different numbers of affected coronary branches. GRACE risk score was calculated for patients with ACS and sCLEC-2 levels in patients with different risk were compared. Logistic regression was used to evaluate plasma sCLEC-2 as an independent risk factor for ACS. Receiver operating curve (ROC) was constructed to analyze the diagnostic value of plasma sCLEC-2 in CHD. The relation of plasma sCLEC-2 and plasma sGPVI as a classical indicator of platelet activation was evaluated using Pearson’s linear regression.Results:Patients with CHD had a significantly higher plasma sCLEC-2 level (181.86 ±146.21 pg/mL vs.121.68 ± 56.24 pg/mL, P<0.001) compared to healthy controls. Subgroup analysis showed that ACS patients had had significantly increased plasma sCLEC-2 levels compared to healthy controls (187.74±151.42 pg/mL vs 121.68±56.24 pg/mL, P<0.001) and non-ACS patients (187.74±151.42 pg/mL vs 126.47±60.98 pg/mL, P<0.05). No significant difference of plasma sCLEC-2 was displayed between non-ACS patients and healthy controls (P>0.05). Plasma sCLEC-2 levels in CAD patients with single, double and triple coronary branch lesions were 196.98±161.44pg/mL,151.73 ±118.79 pg/mL, and 190.29±145.87 pg/mL, without any observed statistical difference (P>0.05). Patients with ACS were stratified as low risk, medium and high risk according to their GRACE scores on admission. Plasma levels of sCLEC2 (Low risk 205.46±185.98 pg/mL, Medium risk 177.83±137.53 pg/mL, High risk 190.37±149.48 pg/mL) were not significantly different among these groups (P>0.05). Logistic regression showed that high plasma sCLEC-2 level (OR= 1.006,95% CI:1.000-1.012, P=0.035) is an independent risk factor for ACS. Receiver operating curve demonstrated that high level of plasma sCLEC-2 can be an effective diagnostic index for CHD, with an AUC of 0.627,95% CI of 0.525～0.728, threshold value of 193.65 pg/mL, sensitivity of 25.3% and specificity of 96.8%(P<0.05). Pearson’s linear regression indicated that plasma sCLEC2 level is positively correlated with plasma sGPVI that is an established marker for platelet activation (r=0.474, P<0.001).Conclusions:Plasma level of sCLEC-2 is significantly increased in patients with CAD. Patients with ACS have higher plasma sCLEC-2 levels compared to non-ACS patients and controls. High plasma sCLEC-2 level is an independent risk factor of ACS. Elevated plasma sCLEC-2 level is an effective diagnostic index for CHD. Plasma level of sCLEC-2 is positively correlated with plasma sGPVI level, rendering it a novel indicator of platelet activation. Part Ⅱ The regulatory mechanism of elevated level plasma soluble CLEC-2Objective:To investigate the causes resulting in the increased plasma level of CLEC-2 in CHD and explore the related mechanisms.Methods:The surface expression of CLEC-2 was determined by flow cytometry analysis. Washed platelets were isolated from the healthy volunteers and stimulated with 50 μg/mL of ox-LDL to detect the mean fluorescence intensity to confirm CLEC-2 shedding. Moreover, the related signaling pathway in platelet CLEC-2 shedding induced by ox-LDL tested in the presence of kinase inhibitor PP2, PRT-060318 and Ro-31-8820. CLEC-2 levels in the supernatants were measure by ELISA when washed platelets were stimulated by the combination of thrombin, oxLDL and 8-pCPT-cGMP. to stimulated the washed platelets and the level of CLEC-2 secretion from platelets was determined by ELISA. The pathways involved in platelet CLEC-2 secretion were determined by modulation of different signals by MnTMPyP, PP2 or Ro-31-8220 when platelet secretion magnification signaling and integrin outside-in signaling were blocked.Results:High expression of CLEC-2 was found in platelet surface rather than dendritic cells, B cells, T cells, monocytes and neutrophils. Ox-LDL stimulation led to CLEC-2 shedding from platelet surface, which was mediated by Src family kinase-Syk-PKC signaling. PKG-cGMP signaling was found to be involved in CLEC-2 secretion from platelets, as well as upstream ROS, Src family kinase and PKC.Conclusions:The elevated plasma level of soluble CLEC-2 in was achieved in 2 ways, including shedding from platelet surface and secretion from platelet granules. These mechanisms are mediate through the Src family kinase-Syk-PKC pathway and Src-PKC-ROS-cGMP signaling, respectively.更多还原