Dickkopf1对人血管平滑肌细胞功能影响及与急性冠脉综合征血清因子的关系

发布时间：2018-04-26 07:40

本文选题：急性冠脉综合征 + 动脉粥样硬化　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景急性冠脉综合征(acute coronary syndrome,ACS)是冠状动脉粥样硬化性心脏病(coronary atherosclerosis heart disease,CAD)中的急性临床事件,包括非ST 段抬高型 ACS(non-ST-segment acute coronary syndrome,NST-ACS)和急性ST 段抬高型心肌梗死(ST-segment elevation myocardial infarction,STEMI),前者又包括不稳定型心绞痛(unstable angina,UA)和急性非ST段抬高型心肌梗死(non-ST-segment elevation myocardial infarction,NSTEMI)。冠状动脉粥样硬化(atherosclerosis,AS)斑块的破裂、溃烂或糜烂引起动脉内血栓的形成以及动脉粥样硬化斑块负荷增加共同成为ACS主要的病理生理机制。氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)是动脉粥样硬化的主要病理因素,可影响血管平滑肌细胞、巨噬细胞、血管内皮细胞等多种细胞的功能。Dickkopfl(DKK1)通过抑制经典Wnt通路参与胚胎发育、多种疾病的发生等生物过程。研究证实,STEMI患者外周血DKK1浓度升高,DKK1可能用于预测ACS患者再次发生不良心血管事件的风险。DKK1还可参与动脉粥样硬化发生过程中血小板与内皮细胞间的炎症反应,介导震荡剪切力作用下血管单核细胞与内皮细胞的粘附功能,参与巨噬细胞对脂质的吞噬和内皮细胞的凋亡等。但目前尚不清楚DKK1是否影响动脉粥样硬化发生发展中血管平滑肌细胞的功能。纤溶酶原激活物抑制剂-1(plasminogen activator inhibitor-1,PAI1)可通过抑制纤维蛋白降解,间接促血栓形成,参员与AMI的病理改变;组织因子(tissue factor,TF)是参与凝血级联反应的重要成员,也可参与AMI患者冠状动脉血栓的形成。白细胞介素-17(interlukin-17,IL-17)和白细胞介素-1β(interlukin-1β,IL-1β)被证实可参与动脉粥样硬化的炎症反应。而DKK1同样作为参与动脉粥样硬化发生发展的分子,其在此过程中与ACS血清因子PAI1、TF、IL-17、IL-1β的相关性如何尚不清楚。目的1.在细胞实验中,观察DKK1对人血管平滑肌细胞功能的影响。2.在临床研究中,探讨DKK1与急性冠脉综合征血清因子PAI1、TF、IL-17、IL-1β的相关性。方法1.细胞实验选择状态良好的8-10代人主动脉平滑肌细胞(human aortic smooth muscle cells,HASMCs)用于实验,设置三组ox-LDL浓度梯度,分别为50、100、150ug/ml,每组ox-LDL刺激HASMCs0、3、6、12、24h后,检测DKK1蛋白或mRNA表达水平。设置 Negative Control(NC)组、NC+ox-LDL 刺激组、DKK1 siRNA 刺激组、DKK1siRNA+ox-LDL刺激组和DKK1过表达组。收集细胞总蛋白,western blot检测 DKK1、collagen Ⅲ、P4Hα1、MMP-2、MMP-9 和 PCNA 的表达水平;EdU增殖实验检测各组HASMCs增殖水平。2.临床实验按照入组标准,收集2017年1月至2017年3月于山东大学齐鲁医院心内科和急诊心内科住院治疗的ACS患者,将其分为UA组和AMI组,以及于健康体检中心查体的正常对照。采集入选者空腹血,并收集完整住院病历或体检信息。用酶联免疫吸附测定法(enzymelinked immunnosorbent assay,ELISA)检测各样本血浆DKK1、PAI1、TF、IL-17和IL-1β的浓度。3.统计方法使用SPSS 17.0软件进行分析:运用单因素方差分析检测多组数据组间差异,Pearson直线相关分析和Spearman直线相关分析检测数据相关性,受试者工作特征曲线(receiver operating characteristic curve,ROC曲线)初步分析诊断敏感度和特异度,显著性水平p值0.05为有统计学差异。结果1.细胞实验结果(1)ox-LDL 上调 HASMCs 中 DKK1 的表达Western blot结果显示,与空白对照相比,100 ug/mlox-LDL刺激HASMCs 12h后,DKK1蛋白表达显著上调(p0.01);RT-PCR结果显示,与空白对照相比,100 ug/ml ox-LDL 刺激 HASMCs 3h 后,DKK1 mRNA 表达增加(p0.05)。(2)DKK1影响HASMCs的胶原代谢Western blot结果显示,与NC组相比,ox-LDL刺激HASMCs或DKK1过表达后,HASMCs中collagen Ⅲ、P4Hα1、MMP-2和MMP-9蛋白表达均上调(p0.05);与NC组相比,siRNA干扰DKK1表达后,MMP-2表表达显著下调(p0.01)。在ox-LDL刺激下,siRNA干扰DKK1的表达可减少collagen Ⅲ、P4Hα1、MMP-2 和 MMP-9 的表达(p0.01)。(3)DKK1影响HASMCs的增殖EdU染色实验显示,与NC组相比,ox-LDL刺激或过表达DKK1可促进HASMCs 增殖(p0.01);干扰 DKK1 的表达,HASMCs 增殖减少(p0.01)。Western blot结果显示,与NC组相比,ox-LDL和DKK1过表达均可诱导HASMCs中PCNA蛋白表达升高(p0.05),抑制DKK1表达后,HASMCs中PCNA表达下调(p0.05)。2.临床研究结果(1)正常对照组、UA组和AMI组的基本信息和血清检查结果组间比较与正常对照组相比,UA组和AMI组患者年龄和血清甘油三酯(TG)、肌酐(Cr)浓度无统计学差异(p0.05),UA组和AMI组患者血清高密度胆固醇脂蛋白(HDL-C)浓度显著降低(p0.05),AMI组谷丙转氨酶(ALT)显著升高(p0.01)。与UA组相比,AMI患者血清总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、ALT、肌酸激酶同工酶(CK-MB)、高敏肌钙蛋白I(CTNI)均升高(p0.05),而 HDL-C 下降(p0.05)。(2)血浆DKK1、PAI1、TF、IL-17和IL-1β浓度的组间比较与正常对照组相比,UA组和AMI组血浆DKK1、PAI1、TF和IL-17浓度均升高(p0.05),IL-1β表达无统计学差异(p0.05)。与UA组相比,AMI组血浆 DKK1、PAI1、TF 和 IL-17 表达增加(p0.05)。(3)血浆DKK1与PAI1、TF、IL-17和IL-1β的相关性分析Pearson直线相关分析显示血浆DKK1与PAI1、TF和IL-17均呈正相关(p0.01)。(4)血浆DKK1用于UA诊断和UA与AMI鉴别诊断特异性、敏感性和临界值的初步分析ROC曲线初步分析血浆DKK1浓度检测用于UA诊断的价值,ROC曲线下面积为0.956,敏感度为0.963,特异度为0.867,临界值为460.60pg/ml。ROC曲线初步分析血浆DKK1浓度检测用于UA和AMI鉴别诊断的价值,ROC曲线下面积为0.869,敏感度为0.947,特异度为0.778,临界值为1200.60pg/ml。结论1.DKK1可参与ox-LDL诱导的血管平滑肌细胞胶原代谢和增殖。2.ACS患者血浆DKK1、PAI1、TF和IL-17的浓度均高于正常对照,其中AMI组较UA组明显升高,血浆DKK1浓度与PAI1、TF、IL-17浓度均呈线性正相关,提示DKK1可能参与ACS发病的病理反应过程。
[Abstract]:Background acute coronary syndrome (acute coronary syndrome, ACS) is an acute clinical event in coronary atherosclerotic heart disease (coronary atherosclerosis heart disease, CAD), including non ST segment elevation ACS (non-ST-segment) and acute segment elevation myocardial infarction. Tion myocardial infarction, STEMI), the former includes unstable angina pectoris (unstable angina, UA) and acute non ST segment elevation myocardial infarction (non-ST-segment elevation myocardial infarction). The rupture of the plaque in the coronary atherosclerosis, ulceration or erosion causes the formation and movement of the thrombus in the arteries. The increasing load of atherosclerotic plaque is the main pathophysiological mechanism of ACS. Oxidized low-density lipoprotein (ox-LDL) is the main pathological factor of atherosclerosis, which can affect the function of vascular smooth muscle cells, macrophages, vascular endothelial cells,.Dickkopfl (DKK1) by inhibiting the function of.Dickkopfl (DKK1). The classical Wnt pathway participates in the biological processes such as embryonic development and the occurrence of various diseases. Studies have shown that the concentration of DKK1 in peripheral blood of STEMI patients is elevated, and DKK1 may be used to predict the risk of adverse cardiovascular events in patients with ACS, and.DKK1 can also be involved in the inflammatory reaction between the platelets and endothelial cells in the process of atherosclerosis and mediate the shock. It is not clear whether DKK1 affects the function of vascular smooth muscle cells in the development and development of atherosclerosis. -1 (plasminogen activator inhibitor-1, plasminogen activator), is not known to be known as the function of vascular smooth muscle cells in the development of atherosclerosis. PAI1) can inhibit fibrin degradation, indirectly promote thrombosis, and the pathological changes of AMI; tissue factor (TF) is an important member of the coagulation cascade reaction, and can also participate in the formation of coronary artery thrombosis in AMI patients. Interleukin -17 (interlukin-17, IL-17) and interleukin -1 beta (Interlukin-1 beta, IL-1 beta) It is proved to be involved in the inflammatory response to atherosclerosis, and DKK1 is also a molecule involved in the development of atherosclerosis. In this process, the correlation with the ACS serum factor PAI1, TF, IL-17, and IL-1 beta is unclear. Objective 1. in cell experiments, the observation of the effect of DKK1 on the function of human vascular smooth muscle cells.2. in clinical study The correlation between DKK1 and the serum factors PAI1, TF, IL-17, and IL-1 beta of acute coronary syndrome was investigated. Methods 1. cells of 8-10 generations of aortic smooth muscle cells (human aortic smooth muscle cells, HASMCs) were selected for the experiment, and three groups of ox-LDL concentration ladder were set up. After 2,24h, the expression level of DKK1 protein or mRNA was detected. Negative Control (NC) group, NC+ox-LDL stimulation group, DKK1 siRNA stimulation group, DKK1siRNA+ox-LDL stimulation group and DKK1 overexpression group were collected. The proliferation level.2. clinical experiment, according to the standard of entry group, collected ACS patients hospitalized in Department of Cardiology and emergency department of cardiology from January 2017 to March 2017 in Qilu Hospital of Shandong University, and divided them into UA group and AMI group, and the normal control in the physical examination center. The fasting blood was collected and the complete hospital records and medical information were collected. Enzymelinked immunnosorbent assay (ELISA) was used to detect the concentration of DKK1, PAI1, TF, IL-17 and IL-1 beta in the plasma of each sample by SPSS 17 software, and the difference between groups of data groups was detected by single factor analysis of variance, Pearson line correlation analysis and linear correlation analysis detection number were detected. According to the correlation, the receiver operating characteristic curve, ROC curve preliminarily analyzed the diagnostic sensitivity and specificity, and the significant level of P value 0.05 was statistically different. Results the results of 1. cell experiment (1) ox-LDL increased DKK1 in HASMCs, Western blot results showed that 100 ug/m compared with the blank control. After lox-LDL stimulation of HASMCs 12h, the expression of DKK1 protein was significantly up-regulated (P0.01). RT-PCR results showed that the expression of DKK1 mRNA increased after 100 ug/ml ox-LDL stimulated HASMCs 3H. (2) The expression of en III, P4H alpha 1, MMP-2 and MMP-9 all up up (P0.05). Compared with the NC group, siRNA interfered DKK1 expression, and the expression of MMP-2 table was significantly down (P0.01). Ox-LDL stimulation or overexpression of DKK1 can promote HASMCs proliferation (P0.01), interference with DKK1 expression, HASMCs proliferation decrease (P0.01).Western blot results, and ox-LDL and DKK1 over expression can induce the increase of protein expression in NC group. Compared with the normal control group, the normal control group and the group UA and the AMI group compared with the normal control group. There was no statistical difference between the age of the UA group and the AMI group, the serum triglyceride (TG) and the creatinine (Cr) concentration (P0.05). The serum high density cholesterol (HDL-C) concentration in the UA and AMI groups decreased significantly (P0.05), and the AMI group cereal ALT significantly increased (P0.01). Compared with group UA, the serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), ALT, creatine kinase isoenzyme (CK-MB) and Gao Min troponin I (CTNI) increased (P0.05), while HDL-C decreased. (2) compared with the normal control group, the concentration of plasma was compared with the normal control group. Plasma levels of DKK1, PAI1, TF and IL-17 increased (P0.05), and there was no significant difference in IL-1 beta expression (P0.05). Compared with the group UA (P0.05), the plasma DKK1, PAI1, and the expression of IL-17 were increased in the AMI group. (3) the correlation analysis between the plasma and the plasma showed that there was a positive correlation between the plasma and the plasma. 0.01) (0.01) (4) a preliminary analysis of the specificity, sensitivity and critical value of plasma DKK1 for the diagnosis of UA and the differential diagnosis of UA and AMI. Preliminary analysis of the value of the ROC curve for the determination of plasma DKK1 concentration for UA diagnosis. The area under the ROC curve is 0.956, the sensitivity is 0.963, the specificity is 0.867, and the critical value is the 460.60pg/ml.ROC curve preliminary analysis of the detection of plasma DKK1 concentration. For the differential diagnosis of UA and AMI, the area under the ROC curve is 0.869, the sensitivity is 0.947, the specificity is 0.778, the critical value is 1200.60pg/ml. conclusion 1.DKK1 can participate in the collagen metabolism of vascular smooth muscle cells induced by ox-LDL and the plasma DKK1, PAI1, TF and IL-17 in.2.ACS patients are higher than those of the normal control, and the AMI group is more obvious than that of the normal control group. Elevated plasma DKK1 concentration was positively correlated with PAI1, TF and IL-17 concentrations, suggesting that DKK1 might be involved in the pathological response process of ACS.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.4

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