差异基因白介素-8在冠心病血瘀证发病过程中的作用研究
|
摘要
本课题组前期应用寡核苷酸基因芯片技术构建了冠心病血瘀证差异基因表达谱,研究结果筛选出IL-8(白介素-8)、FcyRⅢA(免疫球蛋白IgG结晶片段受体Ⅲa)、 PKCβ1(蛋白激酶Cβ1)、HLA-DQB1(主要组织相容性复合体Ⅱβ1)、FOLR3(叶酸受体y3)、PTGDS (前列腺素D2合成酶)等6个目标基因。继而采用基因本体论和显著性通路的分析方法,从分子水平揭示了冠心病血瘀证与炎症免疫反应的相关性,并通过网络拓扑技术分析研究发现差异基因IL-8、FcγRⅢA和PKCβ1在冠心病血瘀证疾病网络中处于关键调控环节。前期研究还发现FcγRⅢA通过介导单核细胞与内皮细胞粘附,影响动脉粥样硬化斑块稳定性从而参与冠心病血瘀证的演变；而对趋化因子IL-8初步研究发现其具有诱导血小板活化的作用,并能协同花生四烯酸和二磷酸腺苷诱导血小板活化,可能是IL-8参与冠心病血瘀证形成的机制之一。
本课题在以往研究基础上对冠心病血瘀证差异基因IL-8进行进一步的临床验证,并对IL-8在冠心病血瘀证形成中的作用进行深入研究分析,结果发现冠心病血瘀证血清IL-8水平明显升高,从蛋白水平证实了IL-8与冠心病血瘀证的相关性。同时研究发现冠心病血瘀证患者的单核细胞血小板聚集率与中性粒细胞血小板聚集率均明显高于冠心病非血瘀证组。基于IL-8具有诱导白细胞膜表面粘附分子MAC-1活化的作用,而MAC-1在白细胞与血小板粘附聚集中发挥重要作用,那么在冠心病血证中白细胞聚集率的升高是否通过IL-8诱导MAC-1活化而发挥作用?本研究通过IL-8诱导外周血中性粒细胞、单核细胞和血小板构建模型,结果发现IL-8能够诱导外周血中性粒细胞和单核细胞聚集,也能够诱导MAC-1活化,且冠心病血瘀证组均明显高于非血瘀证和健康组,采用非选择性拮抗IL-8受体后,中性粒细胞、单核细胞聚集率和MAC-1活化水平均明显下降；同时发现IL-8诱导中性粒细胞血小板聚集和单核细胞血小板聚集后大量表达具有促凝活性的组织因子,并诱导其分泌具有活化血小板功能的组织蛋白酶G、中性粒细胞弹性蛋白酶和血小板活化因子；研究还发现,IL-8除了诱导白细胞活化黏附血小板外,对血小板还有直接活化作用,IL-8受体CXCR1在血小板膜上中量表达,IL-8与该受体特异结合活化血小板,而Reparixin拮抗CXCR1后,血小板聚集率及血小板膜蛋白GPⅡb/Ⅲa表达水平明显下降,就活血化瘀药物对上述生物过程的干预效应进行了研究,发现其具有一定的抑制效应。
本课题研究分为两部分：文献综述和实验研究。
1文献综述：本部分就冠心病血瘀证基因组及蛋白质组学研究进展以及IL-8在冠心病中作用两个方面进行综述。
2实验研究：包括以下四个部分。
研究一冠心病血瘀证患者白介素-8血清学水平及白细胞血小板聚集率测定
目的：探讨冠心病血瘀证与IL-8血清水平、白细胞血小板聚集率及血小板膜蛋白GPⅡb/Ⅲa表达水平的相关性。
方法：冠心病诊断参照1999年美国心脏病学会(ACC)/美国心脏协会(AHA)/美国医师学会及美国内科学会(ACP-ASIM)联合协定关于《慢性稳定性心绞痛诊疗指南》、《不稳定性心绞痛、非ST段抬高心肌梗死诊疗指南》。同时,参考中国中西医结合学会活血化瘀专业委员会制定的血瘀证诊断标准。筛选符合冠心病、血瘀证、健康人入选标准的冠心病血瘀证组、冠心病非血瘀证组和健康对照组分别为53、49、45例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血6mL,前2m1弃用,2m1采用流式细胞术检测白细胞血小板聚集率及血小板膜蛋白GPⅡb/Ⅲa表达水平,另2m1应用酶联免疫吸附法(ELISA)检测IL-8血清水平。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、合并疾病和用药情况方面无显著性差异,具有可比性。
①冠心病IL-8血清水平较健康对照组(47.91±2.93)明显升高,且冠心病血瘀证组(72.58±4.12)较非血瘀证组(58.72±3.90)升高明显。
②冠心病血瘀证组(19.68±0.98)血小板膜蛋白GPⅡb/Ⅲa表达水平较非血瘀证组(9.97±0.85)健康组(8.06±0.34)明显升高,而冠心病非血瘀证组较健康组仅有升高趋势。
③冠心病PLT-M聚集率和PLT-PMN聚集率较健康组(18.87±1.73)显著升高,冠心病血瘀证组(46.91±2.66)较非血瘀证组(31.56±3.17)有升高明显。
结论：血清IL-8水平、血小板膜蛋白GPⅡb/Ⅲa活性、单核细胞血小板聚集率、中性粒细胞血小板聚集率与冠心病血瘀证明显相关。
研究二IL-8对单核细胞、中性粒细胞表面粘附分子及组织因子表达的影响
目的：研究IL-8诱导白细胞血小板聚集机制,探讨IL-8参与冠心病及冠心病血瘀证的发病过程。
方法：纳入冠心病血瘀证患者、冠心病非血瘀患者和健康志愿者各15例为研究对象,冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康志愿者于清晨空腹状态下抽取静脉血6mL,前2m1弃用,置于EDTA抗凝真空采集管中。标本分为4组：空白对照组,35μl纯水；IL-8组,100ng/ml IL-8; Reparixin组,100ng/ml IL-8+1μM Reparixin; fMLP组,1μM fMLP。37℃温育20min,加入0.5%的多聚甲醛固定2ml,2-8℃阴暗处放置30mmin,应用流式细胞术检测各组中性粒细胞和单核细胞的表面粘附分子MAC-1活化水平、血小板与中性粒细胞和单核细胞聚集率、血小板表面组织因子以及血小板与中性粒细胞和单核细胞形成聚集后组织因子的表达水平。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数和合并疾病方面无显著性差异,具有可比性。
①IL-8诱导的粘附分子MAC-1在健康者单核细胞(8.26±2.23)和中性粒细胞(5.93±2.83)表面的表达水平均较空白对照组均明显升高,非选择性拮抗IL-8受体后,中性粒细胞(3.08±2.08)和单核细胞(4.9±1.79)表面的粘附分子MAC-1的表达水平均明显下降。
②冠心病组单核细胞和中性粒细胞表面粘附分子MAC-1表达水平较健康组明显升高,且冠心病血瘀证高于非血瘀证组(P<0.05)。
③IL-8诱导的健康者PLT-PMN聚集率(22.85±3.57)和PLT-M(39.58±6.48)聚集率较空白对照组均明显升高；拮抗IL-8受体后,PLT-PMN(11.84±1.66)和PLT-M(25.41±3.23)聚集率均明显下降。
④冠心病组的PLT-PMN聚集率和PLT-M聚集率较健康组显著升高,且冠心病血瘀证组的PLT-PMN聚集率和PLT-M聚集率均显著高于非血瘀证组(P<0.05)。
⑤IL-8诱导PLT-PMN聚集后组织因子表达检测结果提示,冠心病血瘀证组(34.96±6.15)高于非血瘀证组的表达水平(27.7±5.27),且较同组血小板膜表面表达的组织因子升高更加明显。
结论：
①IL-8可能通过活化中性粒细胞和单核细胞的粘附分子MAC-1促进其与血小板的聚集参与冠心病血瘀证形成。
②IL-8诱导血小板与中性粒细胞、单核细胞聚集后,促使其大量表达具有促凝活性的组织因子,可能是IL-8参与冠心病血瘀证形成的机制之一。
研究三血小板膜IL-8受体CXCR1表达及功能分析
目的：探讨IL-8对血小板的直接活化作用和活化机制。
方法：以5例健康志愿者为研究对象,于清晨空腹状态下抽取静脉血5ml,分离血小板,应用去白细胞滤器纯化血小板,抽提总RNA,应用Real-time RT-PCR技术检测IL-8受体CXCR1基因表达水平；以15例健康志愿者为研究对象,于清晨空腹状态下抽取静脉血,制备PRP和PPP,采用比浊法检测血小板聚集率和流式细胞术检测血小板膜蛋白GPⅡb/Ⅲa的表达。
结果：
①血小板中CXCR1的相对表达量在1.22-2.37之间,呈中度表达,紫外分光光度计检测RNA纯度,质量符合Real-Time RT-PCR实验要求。
②低、中、高不同浓度IL-8对血小板聚集(7.85±1.03,12.56±1.68,19.93±1.52)均有一定的诱导作用,且呈剂量依赖,Reparixin阻断受体后血小板的聚集率明显下降(6.24±1.27)。
③低、中、高不同浓度IL-8刺激后,血小板膜蛋白CD62p的表达(15.46±1.58,28.57±1.64,42.09±2.87)均有不同程度增加,存在量效关系,Reparixin拮抗IL-8受体后,血小板膜蛋白CD62p的表达(17.04±1.98)显著降低。
结论：
①趋化因子IL-8受体CXCR1在血小板膜表面呈中量表达。
②IL-8可通过与血小板膜表面受体CXCR1结合,从而直接活化血小板。
研究四IL-8对人外周血中性粒细胞分泌的影响及活血化瘀中药干预效应的研究
目的：研究IL-8对人外周血中性粒细胞分泌的影响及活血化瘀中药对其干预效应。
方法：分离、鉴定并培养人外周血中性粒细胞,贴壁培养2h得到的中性粒细胞置于全RPMI1640培养基进行如下干预,空白对照组：35μ1培养基；IL-8组：100ng/ml IL-8; Reparixin组：100ng/ml IL-8+25μl Reparixin; fMLP组：1μMfMLP;川芎嗪组：100ng/ml IL-8+20μl川芎嗪；芍药苷组:100ng/ml IL-8+20μl芍药苷。如上干预后各组中性粒细胞继续培养30min,收集细胞上清液,采用酶联免疫吸附法(ELISA)法检测各组Cathepsin G、NE和PAF含量。
结果：
①重组细胞因子IL-8干预30min后中性粒细胞释放的Cathepsin G和NE较空白对照组显著升高(P<0.01)。
②重组细胞因子IL-8干预30min后中性粒细胞释放的PAF较空白对照组明显升高,拮抗受体CXCR1后,PAF的释放水平明显下降。
③川芎嗪干预后,中性粒细胞释放的Cathepsin G和NE较IL-8组显著降低(P<0.05)；芍药苷干预后中性粒细胞释放的PAF明显降低(P<0.05)。
结论：
①重组细胞因子IL-8在体外能刺激人外周中性粒细胞后分泌具有活性血小板和促凝活性的Cathepsin G、NE和PAF,拮抗受体表达后PAF等分泌水平下降。
②活血化瘀中药单体芍药苷和川芎嗪分别对重组细胞因子IL-8诱导中性粒细胞释放Cathepsin G、NE与PAF具有抑制作用,有类受体拮抗效应。
Coronary heart disease (CHD) is the leading cause of death worldwide. Genetic factors and environmental factors are responsible for the development of CHD. The underlying mechanisms of CHD have not yet been elucidated. Previous studies have investigated the differential gene expression profiles in peripheral leukocytes from CHD patients with blood stasis syndrome (BSS) by oligonucleotide microarray technique. We have shown that the immune inflammatory response is correlated to the development of CHD and interleukin-8(IL-8), Fc receptor III A of immunoglobulin G (FcyRIIIA, also named CD16), protein kinase C beta1(PKCβ1), HLA-DQB1, FOLR3, PTGDS were involved in the development of CHD with BSS. Of them, IL-8, FcyRIIIA and PKCβ1played the key role in the regulation of CHD with BSS. In previous studies, It was shown that FcyRIIIA mediated the adhesion of monocytes to endothelial cells and affected the stability of atheromatous plaque in the way of inflammation factors. IL-8induced the activation of platelet by only or synergism with arachidonic acid and Adenosine diphosphate. FcyRIIIA and IL-8may participate in the progress of CHD with BSS in these ways.
We conducted further studies to confirm the correlation of chemokine IL-8to CHD with BSS and its action in the development of CHD with BSS. We investigated the protein expression of IL-8in serum in CHD patients with BSS, CHD patients with non-BSS and the healthy control by enzyme linked immunosorbent assay. There was a significant increase of IL-8at the protein level in CHD patients with BSS. We also investigated the platelet leukocyte aggregate in whole blood in CHD patients with BSS, CHD patients with non-BSS and the healthy control by flow cytometry. There was a remarkable increase of platelet monocyte aggregation and platelet neutrophil aggregation in CHD patients with BSS. Previous study showed that IL-8induced the activation of adhesion molecules MAC-1at the membrane surface of leukocyte and MAC-1plays a role in the platelet leukocyte aggregation. We come up with hypotheses that IL-8could induce the platelet leukocyte aggregation by the activation of adhesion molecules MAC-1in the progress of CHD with BSS.In our study, we observed the level of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation by the model of IL-8induced monocytes, neutrophils and platelet in whole blood of CHD patients with BSS, CHD patients with non-BSS and the healthy control. There were marked elevations in activation of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation after inducing of IL-8in the healthy control. The level of activation of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation in CHD patients with BSS was much higher than those of CHD patients with non-BSS. Meanwhile, there were abundant expression of the tissue factor on the surface of platelet-bound monocytes and platelet-bound neutrophils. Neutrophils also secreted Cathepsin G、NE and PAF, which induce the activation of platelet and have the features of coagulation activity. In addition, IL-8induced the activation of platelet directly after binding with the receptor CXCR1which expressed on the surface of platelet. Moreover, we observed inhibiting effect of activating blood circulation herbs on the above mentioned biological effect of IL-8.
This study is divided into two parts:literature review and experimental research.
1Literature review:Including the following two reviews:Advances in CHD with BSS in Genomics and Proteomics Research and the advances of IL-8in CHD.
2Experimental research:Including the following four parts.
Study I:The determination of serum IL-8and platelet leukocyte aggregation in CHD with BSS.
Objective:To discuss the correlation of serum level of IL-8, the platelet monocyte aggregation and platelet neutrophil aggregation with the CHD with BSS.
Methods:All cases were divided into CHD patients with BSS (53), CHD patients with non-BSS (49) and the healthy control (45). CHD patients were diagnosed according to the1999ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the2002ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least50%was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. Patients with BSS were diagnosed according to guidelines for the diagnosis of BSS. The IL-8in serum was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by enzyme linked immunosorbent assay. The platelet monocyte aggregation, platelet neutrophil aggregation and the expression of GPⅡb/Ⅲa at the surface of platelet were determined in whole blood by flow cytometry.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.
①There was a significant increase of IL-8in serum in CHD, compared to the healthy control (47.91±2.93). Additionally, the level of IL-8in serum in CHD patients with BSS (72.58±4.12) was higher than that in CHD patients with non-BSS (58.72±3.90)
②The expression of GP Ⅱb/Ⅲa at surface of platelet was much increased in CHD patients with BSS (19.68±0.98) than that in CHD patients with non-BSS (9.97±0.85). However, the expression of GP Ⅱb/Ⅲa at surface of platelet had raising trend in CHD patients with non-BSS, compared to the healthy control.
③There were significant increases of platelet monocyte aggregation and platelet neutrophil aggregation in CHD that those in the healthy control. Additionally, the platelet monocyte aggregation and platelet neutrophil aggregation increased remarkably in CHD patients with BSS(46.91±2.66) than those in CHD patients with non-BSS(31.56±3.17).
Conclusion:
The serum level of IL-8, the platelet monocyte aggregation and platelet neutrophil aggregation were markedly correlated to the CHD with BSS.
Study II:The effect of IL-8on the expression of adhesion molecules MAC-1on monocytes and neutrophils, and the expression of tissue factor at the surface on platelet-bound monocytes and platelet-bound neutrophils.
Objective:To study the active mechanism of IL-8induced platelet leukocyte aggregation and the activation of IL-8in the development of CHD with BSS.
Methods:CHD patients were diagnosed according to the1999ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the2002ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least50%was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. All cases were divided into CHD patients with BSS, CHD patients with non-BSSand the healthy control,15in each group. Taken6ml blood and divived into four groups:CON,35μl pure water, IL-8group,100ng/ml IL-8, Reparixin group,100ng/ml IL-8+1μM Reparixin, fMLP group. Then, these were incubating at37℃for20min and added0.5%paraformaldehyde. After30min standing, the adhesion molecules MAC-1, platelet monocyte aggregation, platelet neutrophil aggregation and the expression of tissue factor at its surface.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.
①The expression of adhesion molecules MAC-1at monocytes (8.26±2.23) and neutrophils(5.93±2.83(induced by chemokine IL-8markedly increased than that in CON group in the healthy control. After addition of Reparixin which is a non-selective antagonist to IL-8receptors, the expression of adhesion molecules MAC-1at monocytes (3.08±2.08) and neutrophils (4.9±1.79) significantly decreased in Reparixin group in the healthy control.
②The expression of adhesion molecules MAC-1at monocytes and neutrophils in IL-8group in CHD remarkably increased than those in the healthy control. Additionally, the expression of adhesion molecules MAC-1at monocytes and neutrophils in IL-8group in CHD patients with BSS significantly increased than those in CHD patients with non-BSS (P<0.05).
③The platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group increased remarkably than those in CON group in the healthy control. After addition of Reparixin, the platelet monocyte aggregation and the platelet neutrophil aggregation decreased markedly than those in Reparixin group in the healthy control.
④The platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group in CHD remarkably increased than those in the healthy control. Additionally, the platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group in CHD patients with BSS significantly increased than those in CHD patients with non-BSS (P<0.05).
⑤The expression of tissue factor at the surface of platelet-bound monocytes and platelet-bound neutrophils increased significantly in IL-8group in CHD patients with BSS than those at surface of platelet (P<0.01). Additionally, the expression of tissue factor at the surface of platelet-bound monocytes and platelet-bound neutrophils in IL-8group in CHD patients with BSS increased markedly than those in CHD patients with non-BSS (P<0.05)
Conclusion:The chemokine IL-8may participate in the development of CHD with BSS in the way of inducing the activation of adhesion molecules MAC-1at the surface monocyte and neutrophil, and the formation of platelet monocyte aggregation and platelet neutrophil aggregation.
Study III:The study on expression of IL-8receptor CXCR1at the membrane of platelet and the role of CXCR1in the activation of platelet.
Objective:To study the effect of IL-8on the activation of platelet.
Methods:Human blood platelet from5healthy volunteers was isolated by gradient centrifugation and was purified by the filter which can deplete the leukocytes. The mRNA expression of chemokine IL-8was detected in the way of Real-time RT-PCR. Human blood from15healthy volunteers was prepared for platelet rich plasma (PRP) and platelet poor plasma(PPP). The platelet aggregation was determined by turbidimetry and the expression of membrane protein GPⅡb/Ⅲa at the platelet by the flow cytometry.
Results:
①The relative expression of CXCR1mRNA was between1.22-2.37. The purification of RNA was in accord with experiment requirement of Real-Time RT-PCR.
②IL-8induced the activation of platelet in different concentrations in dose-dependent manner. Reparixin decreased the platelet aggregation and the expression of membrane protein GP II b/IIIa at the platelet, which reverse the effect of CXCRl in the activation of platelet.
Conclusion:
①One of chemokine IL-8receptor CXCR1expressed moderately at the platelet.
②IL-8induced the activation of platelet directly by binding to the CXCR1at the the platelet.
Study IV:Effects of IL-8on the secretion of peripheral blood neutrophils and the intervention effect of active blood herbs on their secretions.
Objective:To study the effects of IL-8on the secretion of peripheral blood neutrophils and the intervention effect of active blood herbs on their secretions.
Methods:Human blood neutrophils was isolated by gradient centrifugation and was cultured in RPMI1640medium for2hours, according to groups as follows:CON group:35μl RPMI1640medium, IL-8group:100ng/ml IL-8, Reparixin group:100ng/ml IL-8+25μl Reparixin, fMLP group:1μMfMLP, Ligustrazine group:100ng/ml IL-8+20μl ligustrazine, Paeoniflorin group:100ng/ml IL-8+20μ plaeoniflorin. After the samples were treated as mentioned above, the human blood neutrophils were cultured for30min and collected the cell culture supernatant. The concentrations of Cathepsin G, NE and PAF were detected by enzyme linked immunosorbent assay.
Results:
①The secretion of Cathepsin G and NE from human blood neutrophils induced by recombinant human cytokines IL-8increased remarkably than those in CON group(P<0.01).
②The secretion of PAF from human blood neutrophils induced by recombinant human cytokines IL-8increased significantly than that in CON group and decreased markedly in Reparixin group than that in IL-8group.
③The secretion of Cathepsin G and NE from human blood neutrophils in Ligustrazine group decreased significantly than those in IL-8group (P<0.05). The secretion of PAF from human blood neutrophils in Paeoniflorin group decreased remarkably than that in IL-8group (P<0.05)
Conclusion:
①IL-8induced the secretion of Cathepsin G, NE and PAF from human blood neutrophils, which induce the activation of platelet and have procoagulant activity.
②The active blood herbs reverse the effects of IL-8on the secretion of Cathepsin G, NE and PAF from human blood neutrophils, which was similar to activation of receptor antagonist.
本课题在以往研究基础上对冠心病血瘀证差异基因IL-8进行进一步的临床验证,并对IL-8在冠心病血瘀证形成中的作用进行深入研究分析,结果发现冠心病血瘀证血清IL-8水平明显升高,从蛋白水平证实了IL-8与冠心病血瘀证的相关性。同时研究发现冠心病血瘀证患者的单核细胞血小板聚集率与中性粒细胞血小板聚集率均明显高于冠心病非血瘀证组。基于IL-8具有诱导白细胞膜表面粘附分子MAC-1活化的作用,而MAC-1在白细胞与血小板粘附聚集中发挥重要作用,那么在冠心病血证中白细胞聚集率的升高是否通过IL-8诱导MAC-1活化而发挥作用?本研究通过IL-8诱导外周血中性粒细胞、单核细胞和血小板构建模型,结果发现IL-8能够诱导外周血中性粒细胞和单核细胞聚集,也能够诱导MAC-1活化,且冠心病血瘀证组均明显高于非血瘀证和健康组,采用非选择性拮抗IL-8受体后,中性粒细胞、单核细胞聚集率和MAC-1活化水平均明显下降；同时发现IL-8诱导中性粒细胞血小板聚集和单核细胞血小板聚集后大量表达具有促凝活性的组织因子,并诱导其分泌具有活化血小板功能的组织蛋白酶G、中性粒细胞弹性蛋白酶和血小板活化因子；研究还发现,IL-8除了诱导白细胞活化黏附血小板外,对血小板还有直接活化作用,IL-8受体CXCR1在血小板膜上中量表达,IL-8与该受体特异结合活化血小板,而Reparixin拮抗CXCR1后,血小板聚集率及血小板膜蛋白GPⅡb/Ⅲa表达水平明显下降,就活血化瘀药物对上述生物过程的干预效应进行了研究,发现其具有一定的抑制效应。
本课题研究分为两部分：文献综述和实验研究。
1文献综述：本部分就冠心病血瘀证基因组及蛋白质组学研究进展以及IL-8在冠心病中作用两个方面进行综述。
2实验研究：包括以下四个部分。
研究一冠心病血瘀证患者白介素-8血清学水平及白细胞血小板聚集率测定
目的：探讨冠心病血瘀证与IL-8血清水平、白细胞血小板聚集率及血小板膜蛋白GPⅡb/Ⅲa表达水平的相关性。
方法：冠心病诊断参照1999年美国心脏病学会(ACC)/美国心脏协会(AHA)/美国医师学会及美国内科学会(ACP-ASIM)联合协定关于《慢性稳定性心绞痛诊疗指南》、《不稳定性心绞痛、非ST段抬高心肌梗死诊疗指南》。同时,参考中国中西医结合学会活血化瘀专业委员会制定的血瘀证诊断标准。筛选符合冠心病、血瘀证、健康人入选标准的冠心病血瘀证组、冠心病非血瘀证组和健康对照组分别为53、49、45例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血6mL,前2m1弃用,2m1采用流式细胞术检测白细胞血小板聚集率及血小板膜蛋白GPⅡb/Ⅲa表达水平,另2m1应用酶联免疫吸附法(ELISA)检测IL-8血清水平。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、合并疾病和用药情况方面无显著性差异,具有可比性。
①冠心病IL-8血清水平较健康对照组(47.91±2.93)明显升高,且冠心病血瘀证组(72.58±4.12)较非血瘀证组(58.72±3.90)升高明显。
②冠心病血瘀证组(19.68±0.98)血小板膜蛋白GPⅡb/Ⅲa表达水平较非血瘀证组(9.97±0.85)健康组(8.06±0.34)明显升高,而冠心病非血瘀证组较健康组仅有升高趋势。
③冠心病PLT-M聚集率和PLT-PMN聚集率较健康组(18.87±1.73)显著升高,冠心病血瘀证组(46.91±2.66)较非血瘀证组(31.56±3.17)有升高明显。
结论：血清IL-8水平、血小板膜蛋白GPⅡb/Ⅲa活性、单核细胞血小板聚集率、中性粒细胞血小板聚集率与冠心病血瘀证明显相关。
研究二IL-8对单核细胞、中性粒细胞表面粘附分子及组织因子表达的影响
目的：研究IL-8诱导白细胞血小板聚集机制,探讨IL-8参与冠心病及冠心病血瘀证的发病过程。
方法：纳入冠心病血瘀证患者、冠心病非血瘀患者和健康志愿者各15例为研究对象,冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康志愿者于清晨空腹状态下抽取静脉血6mL,前2m1弃用,置于EDTA抗凝真空采集管中。标本分为4组：空白对照组,35μl纯水；IL-8组,100ng/ml IL-8; Reparixin组,100ng/ml IL-8+1μM Reparixin; fMLP组,1μM fMLP。37℃温育20min,加入0.5%的多聚甲醛固定2ml,2-8℃阴暗处放置30mmin,应用流式细胞术检测各组中性粒细胞和单核细胞的表面粘附分子MAC-1活化水平、血小板与中性粒细胞和单核细胞聚集率、血小板表面组织因子以及血小板与中性粒细胞和单核细胞形成聚集后组织因子的表达水平。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数和合并疾病方面无显著性差异,具有可比性。
①IL-8诱导的粘附分子MAC-1在健康者单核细胞(8.26±2.23)和中性粒细胞(5.93±2.83)表面的表达水平均较空白对照组均明显升高,非选择性拮抗IL-8受体后,中性粒细胞(3.08±2.08)和单核细胞(4.9±1.79)表面的粘附分子MAC-1的表达水平均明显下降。
②冠心病组单核细胞和中性粒细胞表面粘附分子MAC-1表达水平较健康组明显升高,且冠心病血瘀证高于非血瘀证组(P<0.05)。
③IL-8诱导的健康者PLT-PMN聚集率(22.85±3.57)和PLT-M(39.58±6.48)聚集率较空白对照组均明显升高；拮抗IL-8受体后,PLT-PMN(11.84±1.66)和PLT-M(25.41±3.23)聚集率均明显下降。
④冠心病组的PLT-PMN聚集率和PLT-M聚集率较健康组显著升高,且冠心病血瘀证组的PLT-PMN聚集率和PLT-M聚集率均显著高于非血瘀证组(P<0.05)。
⑤IL-8诱导PLT-PMN聚集后组织因子表达检测结果提示,冠心病血瘀证组(34.96±6.15)高于非血瘀证组的表达水平(27.7±5.27),且较同组血小板膜表面表达的组织因子升高更加明显。
结论：
①IL-8可能通过活化中性粒细胞和单核细胞的粘附分子MAC-1促进其与血小板的聚集参与冠心病血瘀证形成。
②IL-8诱导血小板与中性粒细胞、单核细胞聚集后,促使其大量表达具有促凝活性的组织因子,可能是IL-8参与冠心病血瘀证形成的机制之一。
研究三血小板膜IL-8受体CXCR1表达及功能分析
目的：探讨IL-8对血小板的直接活化作用和活化机制。
方法：以5例健康志愿者为研究对象,于清晨空腹状态下抽取静脉血5ml,分离血小板,应用去白细胞滤器纯化血小板,抽提总RNA,应用Real-time RT-PCR技术检测IL-8受体CXCR1基因表达水平；以15例健康志愿者为研究对象,于清晨空腹状态下抽取静脉血,制备PRP和PPP,采用比浊法检测血小板聚集率和流式细胞术检测血小板膜蛋白GPⅡb/Ⅲa的表达。
结果：
①血小板中CXCR1的相对表达量在1.22-2.37之间,呈中度表达,紫外分光光度计检测RNA纯度,质量符合Real-Time RT-PCR实验要求。
②低、中、高不同浓度IL-8对血小板聚集(7.85±1.03,12.56±1.68,19.93±1.52)均有一定的诱导作用,且呈剂量依赖,Reparixin阻断受体后血小板的聚集率明显下降(6.24±1.27)。
③低、中、高不同浓度IL-8刺激后,血小板膜蛋白CD62p的表达(15.46±1.58,28.57±1.64,42.09±2.87)均有不同程度增加,存在量效关系,Reparixin拮抗IL-8受体后,血小板膜蛋白CD62p的表达(17.04±1.98)显著降低。
结论：
①趋化因子IL-8受体CXCR1在血小板膜表面呈中量表达。
②IL-8可通过与血小板膜表面受体CXCR1结合,从而直接活化血小板。
研究四IL-8对人外周血中性粒细胞分泌的影响及活血化瘀中药干预效应的研究
目的：研究IL-8对人外周血中性粒细胞分泌的影响及活血化瘀中药对其干预效应。
方法：分离、鉴定并培养人外周血中性粒细胞,贴壁培养2h得到的中性粒细胞置于全RPMI1640培养基进行如下干预,空白对照组：35μ1培养基；IL-8组：100ng/ml IL-8; Reparixin组：100ng/ml IL-8+25μl Reparixin; fMLP组：1μMfMLP;川芎嗪组：100ng/ml IL-8+20μl川芎嗪；芍药苷组:100ng/ml IL-8+20μl芍药苷。如上干预后各组中性粒细胞继续培养30min,收集细胞上清液,采用酶联免疫吸附法(ELISA)法检测各组Cathepsin G、NE和PAF含量。
结果：
①重组细胞因子IL-8干预30min后中性粒细胞释放的Cathepsin G和NE较空白对照组显著升高(P<0.01)。
②重组细胞因子IL-8干预30min后中性粒细胞释放的PAF较空白对照组明显升高,拮抗受体CXCR1后,PAF的释放水平明显下降。
③川芎嗪干预后,中性粒细胞释放的Cathepsin G和NE较IL-8组显著降低(P<0.05)；芍药苷干预后中性粒细胞释放的PAF明显降低(P<0.05)。
结论：
①重组细胞因子IL-8在体外能刺激人外周中性粒细胞后分泌具有活性血小板和促凝活性的Cathepsin G、NE和PAF,拮抗受体表达后PAF等分泌水平下降。
②活血化瘀中药单体芍药苷和川芎嗪分别对重组细胞因子IL-8诱导中性粒细胞释放Cathepsin G、NE与PAF具有抑制作用,有类受体拮抗效应。
Coronary heart disease (CHD) is the leading cause of death worldwide. Genetic factors and environmental factors are responsible for the development of CHD. The underlying mechanisms of CHD have not yet been elucidated. Previous studies have investigated the differential gene expression profiles in peripheral leukocytes from CHD patients with blood stasis syndrome (BSS) by oligonucleotide microarray technique. We have shown that the immune inflammatory response is correlated to the development of CHD and interleukin-8(IL-8), Fc receptor III A of immunoglobulin G (FcyRIIIA, also named CD16), protein kinase C beta1(PKCβ1), HLA-DQB1, FOLR3, PTGDS were involved in the development of CHD with BSS. Of them, IL-8, FcyRIIIA and PKCβ1played the key role in the regulation of CHD with BSS. In previous studies, It was shown that FcyRIIIA mediated the adhesion of monocytes to endothelial cells and affected the stability of atheromatous plaque in the way of inflammation factors. IL-8induced the activation of platelet by only or synergism with arachidonic acid and Adenosine diphosphate. FcyRIIIA and IL-8may participate in the progress of CHD with BSS in these ways.
We conducted further studies to confirm the correlation of chemokine IL-8to CHD with BSS and its action in the development of CHD with BSS. We investigated the protein expression of IL-8in serum in CHD patients with BSS, CHD patients with non-BSS and the healthy control by enzyme linked immunosorbent assay. There was a significant increase of IL-8at the protein level in CHD patients with BSS. We also investigated the platelet leukocyte aggregate in whole blood in CHD patients with BSS, CHD patients with non-BSS and the healthy control by flow cytometry. There was a remarkable increase of platelet monocyte aggregation and platelet neutrophil aggregation in CHD patients with BSS. Previous study showed that IL-8induced the activation of adhesion molecules MAC-1at the membrane surface of leukocyte and MAC-1plays a role in the platelet leukocyte aggregation. We come up with hypotheses that IL-8could induce the platelet leukocyte aggregation by the activation of adhesion molecules MAC-1in the progress of CHD with BSS.In our study, we observed the level of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation by the model of IL-8induced monocytes, neutrophils and platelet in whole blood of CHD patients with BSS, CHD patients with non-BSS and the healthy control. There were marked elevations in activation of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation after inducing of IL-8in the healthy control. The level of activation of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation in CHD patients with BSS was much higher than those of CHD patients with non-BSS. Meanwhile, there were abundant expression of the tissue factor on the surface of platelet-bound monocytes and platelet-bound neutrophils. Neutrophils also secreted Cathepsin G、NE and PAF, which induce the activation of platelet and have the features of coagulation activity. In addition, IL-8induced the activation of platelet directly after binding with the receptor CXCR1which expressed on the surface of platelet. Moreover, we observed inhibiting effect of activating blood circulation herbs on the above mentioned biological effect of IL-8.
This study is divided into two parts:literature review and experimental research.
1Literature review:Including the following two reviews:Advances in CHD with BSS in Genomics and Proteomics Research and the advances of IL-8in CHD.
2Experimental research:Including the following four parts.
Study I:The determination of serum IL-8and platelet leukocyte aggregation in CHD with BSS.
Objective:To discuss the correlation of serum level of IL-8, the platelet monocyte aggregation and platelet neutrophil aggregation with the CHD with BSS.
Methods:All cases were divided into CHD patients with BSS (53), CHD patients with non-BSS (49) and the healthy control (45). CHD patients were diagnosed according to the1999ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the2002ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least50%was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. Patients with BSS were diagnosed according to guidelines for the diagnosis of BSS. The IL-8in serum was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by enzyme linked immunosorbent assay. The platelet monocyte aggregation, platelet neutrophil aggregation and the expression of GPⅡb/Ⅲa at the surface of platelet were determined in whole blood by flow cytometry.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.
①There was a significant increase of IL-8in serum in CHD, compared to the healthy control (47.91±2.93). Additionally, the level of IL-8in serum in CHD patients with BSS (72.58±4.12) was higher than that in CHD patients with non-BSS (58.72±3.90)
②The expression of GP Ⅱb/Ⅲa at surface of platelet was much increased in CHD patients with BSS (19.68±0.98) than that in CHD patients with non-BSS (9.97±0.85). However, the expression of GP Ⅱb/Ⅲa at surface of platelet had raising trend in CHD patients with non-BSS, compared to the healthy control.
③There were significant increases of platelet monocyte aggregation and platelet neutrophil aggregation in CHD that those in the healthy control. Additionally, the platelet monocyte aggregation and platelet neutrophil aggregation increased remarkably in CHD patients with BSS(46.91±2.66) than those in CHD patients with non-BSS(31.56±3.17).
Conclusion:
The serum level of IL-8, the platelet monocyte aggregation and platelet neutrophil aggregation were markedly correlated to the CHD with BSS.
Study II:The effect of IL-8on the expression of adhesion molecules MAC-1on monocytes and neutrophils, and the expression of tissue factor at the surface on platelet-bound monocytes and platelet-bound neutrophils.
Objective:To study the active mechanism of IL-8induced platelet leukocyte aggregation and the activation of IL-8in the development of CHD with BSS.
Methods:CHD patients were diagnosed according to the1999ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the2002ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least50%was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. All cases were divided into CHD patients with BSS, CHD patients with non-BSSand the healthy control,15in each group. Taken6ml blood and divived into four groups:CON,35μl pure water, IL-8group,100ng/ml IL-8, Reparixin group,100ng/ml IL-8+1μM Reparixin, fMLP group. Then, these were incubating at37℃for20min and added0.5%paraformaldehyde. After30min standing, the adhesion molecules MAC-1, platelet monocyte aggregation, platelet neutrophil aggregation and the expression of tissue factor at its surface.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.
①The expression of adhesion molecules MAC-1at monocytes (8.26±2.23) and neutrophils(5.93±2.83(induced by chemokine IL-8markedly increased than that in CON group in the healthy control. After addition of Reparixin which is a non-selective antagonist to IL-8receptors, the expression of adhesion molecules MAC-1at monocytes (3.08±2.08) and neutrophils (4.9±1.79) significantly decreased in Reparixin group in the healthy control.
②The expression of adhesion molecules MAC-1at monocytes and neutrophils in IL-8group in CHD remarkably increased than those in the healthy control. Additionally, the expression of adhesion molecules MAC-1at monocytes and neutrophils in IL-8group in CHD patients with BSS significantly increased than those in CHD patients with non-BSS (P<0.05).
③The platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group increased remarkably than those in CON group in the healthy control. After addition of Reparixin, the platelet monocyte aggregation and the platelet neutrophil aggregation decreased markedly than those in Reparixin group in the healthy control.
④The platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group in CHD remarkably increased than those in the healthy control. Additionally, the platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group in CHD patients with BSS significantly increased than those in CHD patients with non-BSS (P<0.05).
⑤The expression of tissue factor at the surface of platelet-bound monocytes and platelet-bound neutrophils increased significantly in IL-8group in CHD patients with BSS than those at surface of platelet (P<0.01). Additionally, the expression of tissue factor at the surface of platelet-bound monocytes and platelet-bound neutrophils in IL-8group in CHD patients with BSS increased markedly than those in CHD patients with non-BSS (P<0.05)
Conclusion:The chemokine IL-8may participate in the development of CHD with BSS in the way of inducing the activation of adhesion molecules MAC-1at the surface monocyte and neutrophil, and the formation of platelet monocyte aggregation and platelet neutrophil aggregation.
Study III:The study on expression of IL-8receptor CXCR1at the membrane of platelet and the role of CXCR1in the activation of platelet.
Objective:To study the effect of IL-8on the activation of platelet.
Methods:Human blood platelet from5healthy volunteers was isolated by gradient centrifugation and was purified by the filter which can deplete the leukocytes. The mRNA expression of chemokine IL-8was detected in the way of Real-time RT-PCR. Human blood from15healthy volunteers was prepared for platelet rich plasma (PRP) and platelet poor plasma(PPP). The platelet aggregation was determined by turbidimetry and the expression of membrane protein GPⅡb/Ⅲa at the platelet by the flow cytometry.
Results:
①The relative expression of CXCR1mRNA was between1.22-2.37. The purification of RNA was in accord with experiment requirement of Real-Time RT-PCR.
②IL-8induced the activation of platelet in different concentrations in dose-dependent manner. Reparixin decreased the platelet aggregation and the expression of membrane protein GP II b/IIIa at the platelet, which reverse the effect of CXCRl in the activation of platelet.
Conclusion:
①One of chemokine IL-8receptor CXCR1expressed moderately at the platelet.
②IL-8induced the activation of platelet directly by binding to the CXCR1at the the platelet.
Study IV:Effects of IL-8on the secretion of peripheral blood neutrophils and the intervention effect of active blood herbs on their secretions.
Objective:To study the effects of IL-8on the secretion of peripheral blood neutrophils and the intervention effect of active blood herbs on their secretions.
Methods:Human blood neutrophils was isolated by gradient centrifugation and was cultured in RPMI1640medium for2hours, according to groups as follows:CON group:35μl RPMI1640medium, IL-8group:100ng/ml IL-8, Reparixin group:100ng/ml IL-8+25μl Reparixin, fMLP group:1μMfMLP, Ligustrazine group:100ng/ml IL-8+20μl ligustrazine, Paeoniflorin group:100ng/ml IL-8+20μ plaeoniflorin. After the samples were treated as mentioned above, the human blood neutrophils were cultured for30min and collected the cell culture supernatant. The concentrations of Cathepsin G, NE and PAF were detected by enzyme linked immunosorbent assay.
Results:
①The secretion of Cathepsin G and NE from human blood neutrophils induced by recombinant human cytokines IL-8increased remarkably than those in CON group(P<0.01).
②The secretion of PAF from human blood neutrophils induced by recombinant human cytokines IL-8increased significantly than that in CON group and decreased markedly in Reparixin group than that in IL-8group.
③The secretion of Cathepsin G and NE from human blood neutrophils in Ligustrazine group decreased significantly than those in IL-8group (P<0.05). The secretion of PAF from human blood neutrophils in Paeoniflorin group decreased remarkably than that in IL-8group (P<0.05)
Conclusion:
①IL-8induced the secretion of Cathepsin G, NE and PAF from human blood neutrophils, which induce the activation of platelet and have procoagulant activity.
②The active blood herbs reverse the effects of IL-8on the secretion of Cathepsin G, NE and PAF from human blood neutrophils, which was similar to activation of receptor antagonist.
引文
1.李瑶.基因芯片技术解码生命.北京化学工业出版社,2004,177-198.
2. Tyers, M.,& Mann,M. From genomics to proteomics.Nature,2003,422:193-197.
3. Mahesh Uttamchandani, Junqi Li, Hongyan Sun, et al. Yao. Activity-Based Protein Profiling:New Developments and Directions in Functional Proteomics. ChemBioChem, 2008,9:667-675.
4. Thomas Kocher, Giulio Superti-Furga.Mass spectrometry-based functional proteomics: from molecular machines to protein networks. Nature methods,2007,4(10):807-815.
5.杨保林,王阶,姜燕.反向Northern筛查冠心病血瘀证差异片段.中国中医基础医学杂志,2004,10(9)：669-674.
6.马晓娟,殷惠军,陈可冀.血瘀证患者差异基因表达谱研究.中西医结合报,2004,6(4)：355-360.
7.赵慧辉,王伟,郭淑贞.冠心病不稳定型心绞痛血瘀证的蛋白质组学.中国动脉硬化杂志,2008,16(7)：545-548.
8.吴红金,马增春,高月,等.蛋白质组学技术对冠心病血瘀证相关蛋白的研究.中西医结合心脑血管病杂志2005,3(3)189-191.
9.周倩倩,李治国,黄力.冠心病血瘀证的血清差异蛋白质组学研究.微循环学杂志,2012,22(1)：13-16.
10.刘剑刚,徐浩,董国菊,等.血府逐瘀口服液对冠心病心绞痛患者血小板活化分子表达的影响.长春中医药大学学报,2007,23：29-31.
11. Chen KJ, Xue M, Yin H J. The relationship between platelet activation and coronary heart disease and blood stasis syndrome. Shou Du Yi Ke Da Xue Xue Bao.2008,29(3): 266-269.
12.吴锦,陈可冀,裴正礼,等.冠心病血瘀证患者血小板扫描电镜形态观察.中国医药学报,1990,5(1)：18-20.
13.杨军辉,周小青.冠心病心绞痛血瘀证四亚型与血小板活性相关性探讨.中国中医药咨讯,2011,3(7)：306-307.
14.李雪峰,蒋跃绒,高铸烨,等.冠心病血瘀证血小板差异功能蛋白筛选、鉴定及功能分析.中国中西医结合杂志,2010,30(5)：467-473.
15.殷惠军,马晓娟,蒋跃绒,等.冠心病差异基因表达谱的构建及目标基因的功能分析.科学通报,2009,54(3)：354-359.
16. Ye Huang, Huijun Yin, Jingshang Wang, et al. Aberrant expression of Fc γRIIIA (CD16) contributes to the development of atherosclerosis. Gene.2012,498(l):91-95.
17. LIU Yue, YIN Huijun, JIANG Yuerong, et al. Correlation between platelet gelsolin level and different types of coronary heart disease. Chin Sci Bull,2012,57(6):631-638.
1. Remick GD. Interleukin-8. Crit Care Med,2005,33:s646-s647.
2. Waugh JJD, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res,2008, 14:6735-6741.
3. Standiford TJ, Kunkel SL, Basha MA, et al. Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung. J Clin Invest,1990,86:1945-1953.
4. Gimbrone MA Jr, Obin MS, Brock AF, et al. Endothelial interleukin-8:a novel inhibitor of leukocyte-endothelial interactions. Science,1989,246:1601-1603.
5. RoseMarie Stillie, Shukkur Muhammed Farooq, John R. Gordon, et al.The functional significance behind expressing two IL-8 receptor types on PMN.Journal of Leukocyte Biology,2009,86:529-543.
6. DeForge LE, Fantone JC, Kenney JS, et al. Oxygen radical scavengers selectively inhibit interleukin-8 production in human whole blood. J Clin Invest,1992,90: 2123-2129.
7. Rus HG, Vlaicu R, Niculescu F. Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall. Atherosclerosis,1996,127:263-271.
8. Apostolopoulos J, Davenport P, Tipping PG. Interleukin-8 production by macrophages from atheromatous plaques. Arterioscler Thromb Vase Biol,1996, 16:1007-1012.
9. Geisel J, Jodden V, Obeid R, et al. Stimulatory effect of homocysteine on interleukin-8 expression in human endothelial cells. Clin Chem Lab Med,2003, 41(8):1045-1048.
10. Dje N Guessan P, Riediger F, Vardarova K, et al. Statins control oxidized LDL-mediated histone modifications and gene expression in cultured human endothelial cells. Arterioscler Thromb Vasc Biol,2009,29:380-386.
11. Ryoo SW, Kim DU, Won M, et al. Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovasc Res,2004,62 (1):185-193.
12. Wang YS, Li XJ, Zhao WO. TREM-1 is a positive regulator of TNF-a and IL-8 production in U937 foam cells. Bosn J Basic Med Sci,2012,12(2):94-101.
13. Al-Bannawi A, Al-Wesebai K, Taha S, et al. Chlamydia pneumoniae induces chemokine expression by platelets in patients with atherosclerosis. Med Princ Pract, 2011,20(5):438-443.
14. Deng S, Jepsen S, Dommisch H, et al. Cysteine proteases from Porphyromonas gingivalis and TLR ligands synergistically induce the synthesis of the cytokine IL-8 in human artery endothelial cells. Arch Oral Bio,2011,56(12):1583-1591.
15. Gerszten RE, Garcia-Zepeda EA, Lim YC, et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature,1999, 398:718-723.
16. Koch AE, Volin MV, Woods JM, et al. Regulation of angiogenesis by the C-X-C chemokines interleukin-8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint. Arthritis Rheum,2001,44(1):31-40.
17. Strieter RM, Kunkel SL, Elner VM, et al. Interleukin-8. A corneal factor that induces neovascularization. Am J Pathol,1992,141(6):1279-1284.
18. Arenberg DA, Kunkel SL, Polverini PJ, et al. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J Clin Invest,1996, 12:2792-2802.
19. Simonini A, Moscucci M, Muller DW, et al. IL-8 is an angiogenic factor in human coronary atherectomy tissue. Circulation,2000,101:1519-1526.
20. Li A, Dubey S, Varney ML, et al. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol,2003,170(6):3369-3376.
21. Kyriakakis E, Cavallari M, Pfaff D, et al. IL-8-mediated angiogenic responses of endothelial cells to lipid antigen activation of iNKT cells depend on EGFR transactivation. J Leukoc Biol,2011,90(5):929-939.
22. Luca M, Huang S, Gershenwald JE, et al. Expression of interieukin-8 by human melanoma cells up-regulates MMP-2 activity and increases tumor growth and metastasis. Am J Pathol,1997,151:1105-1113.
23. Moreau M, Brocheriou I, Petit L, et al. Interleukin-8 mediates downregulation of tissue inhibitor of metalloproteinase-1 expression in cholesterol-loaded human macrophages:relevance to stability of atherosclerotic plaque. Circulation,1999, 99:420-426.
24. Teruo Inoue, Hiroshi Komoda, Masako Nonaka, et al. Interleukin-8 as an independent predictor of long-term clinical outcome in patients with coronary artery disease. International Journal of Cardiology,2008,124(3):319-325.
25. Herder C,Baumert J,Thorand B,et al. Chemokines and incident coronary heart disease:results from the MONICA/KORA Augsburg case-cohort study,1984-2002. Arterioscler Thromb Vasc Biol,2006,9:2147-2152.
26. Elmas E, Lang S, Dempfle CE, et al. High plasma levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and interleukin-8 (IL-8) characterize patients prone to ventricular fibrillation complicating myocardial infarction. Clin Chem Lab Med,2007, 45(10):1360-1365.
27. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez M, et al. Prognostic value of interleukin-8 as a predictor of heart failure in patients with myocardial infarction and percutaneous intervention. International Journal of Cardiology,2006,111(1):158-160.
28. De Gennaro L, Brunetti ND, Montrone D, et al. Subacute inflammatory activation in subjects with acute coronary syndrome and left ventricular dysfunction. Inflammation,2012,35(1):363-370.
29. Damas JK, Eiken HG, Oie E, et al. Myocardial expression of CC-and CXC-chemokines and their receptors in human end-stage heart failure. Cardiovasc Res,2000,47(4):778-787.
30. Leonard DA, Merhige ME, Williams BA, et al. Elevated expression of the interleukin-8 receptors CXCR1 and CXCR2 in peripheral blood cells in obstructive coronary artery disease. Coron Artery Dis,2011,22(7):49-49
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Management of Patients with Chronic Stable Angina). J Am Coll Cardiol.1999,33 (7):2092-2197.
2. Braunwald E, Antman EM, Beasley JW, et al. ACC/AHA 2002 guideline updatefor the management of patients with unstable angina and non-ST-segment elevation myocardial infarction-summary article:a report of the American College of Cardiology/American Heart Association task force on practice guidelines (Committee on the Management of Patients with Unstable Angina). J Am Coll Cardiol.2002,40 (7):1366-1374.
3. 卫生部心血管防治研究中心.中国心血管病报告2011.北京：中国大百科全书出版社,2012.
4. Chierchia SL. Inflammation and acute coronary syndromes. Rev Esp Cardiol, 2001,54(10):1135-1140.
5. Li N, Hu H, Lindqvist M, et al. Platelet-leukocyte cross talk in whole blood. Arterioscler Thromb Vasc Biol.2000,20(12):2702-2708.
6. Ruggeri ZM. Platelets in atherothrombosis. Nat Med.2002,8:1227-1234.
7. Gawaz M. Do platelets trigger atherosclerosis? Thromb Haemost.2003, 90:971-972.
8. 李家增,贺石林,王鸿利.血栓病学.北京：科学出版社,1998.P25-27.
9. Picker SM. Platelet function in ischemic heart disease. J Cardiovasc Pharmacol. 2013,61(2):166-174.
10. Weber C. Platelets and chemokines in atherosclerosis:partners in crime. Circ Res.2005,96(6):612-616.
11. Ross R. Atherosclerosis-an inflammatory disease. N Eng J Med.1999, 340:115-126.
12. Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation.2001,104:365-372.
13. Biasucci LM, D Onofrio G, Liuzzo G, et al. Intracellular neutrophil myeloperoxidase is reduced in unstable angina and myocardial infarction, but its reduction is not related to ischemia. J Am Coll Cardiol.1996,27:611-616.
14. Home BD, Anderson JL, John JM et al. Intermountain Heart Collaborative Study Group. Which white blood cell subtypes predict increased cardiovascular risk?. J Am Coll Cardiol.2005,45:1638-1643.
15. Gillum RF, Mussolino ME, Madans JH. Counts of Neutrophils, Lymphocytes, and Monocytes, Cause-specific Mortality and Coronary Heart Disease:The NHANES-I Epidemiologic Follow-up Study. Ann Epidemiol.2005,15:266-271.
16. Bharadwaj D, Stein MP, Volzer M, et al.The major recept or for C- reactive protein on leukocytes is fcgamma receptor. J Exp Med.1999,190(4):585-590.
17. Ott I, Neumann FJ, Kenngott S, et al. Procoagulant inflammatory responses of monocytes after direct balloon angioplasty in acute myocardial infarction. Am J Cardiol.1998,82:938-942.
18. Engler RL, Schmid-Schonbein GW, Pavelec RS. Leokocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol.1983,111:98-111.
19. Zatta A, Prosdocimi M, Bertere V, et al. Inhibition of platelet function by polymorphonuclear leukocytes. J Lab Clin Med.1990,116:651-660.
20. Pidard D, Renesto P, Rahbi S, et al. Regulation of the structure and activity of platelet adhesion receptors by leukocyte proteinases. Nouv Rev Fr Hematol.1994, 36:S99-101.
21. Neuman FJ, Ott I, Marx N, et al. Effect of human recombinant interleukin-6 and interleukin-8 on monocyte procoagulant activity. Arterioscler Thromb Vasc Biol. 1997,17:3399-3405.
22.陈可冀.活血化瘀研究与临床[M]北京：中国协和医科大学,北京医科大学联合出版社,1993,13-17.
23.翁维良.冠心病血瘀证的研究进展.中医杂志.1987,6：464.
24. Habazettl H, Hanusch P, Kupatt C. Effects of endothelium leukocytes platelet interaction on myocardial ischemia-reperfusion injury. Z Kardiol.2000,89 (Supp 19):Ⅸ92-95.
25. Wang J, Zhang S, Jin Y, et al. Elevated levels of platelet monocyte aggregates and related circulating biomarkers in patients with acute coronary syndrome.Int J Cardiol.2007;115(3):361-365.
26. Field EJ, MacLeod I. Platelet adherence to neutrophils. Br Med J.1963, 2:388-389.
27. Konstantopoulos K, Neelamegham S, Burns AR, et al. Venous levels of shear support neutrophil platelet adhesion and neutrophil aggregation in blood via P-selectin and β2-integrin. Circulation.1998,98:873-882.
28. E ppihim er M J, S chaub RG. P-S elect in-dependent inhibit ion of thrombosis during venous stasis. Arterioscler Thromb Vasc B io.2000,20:2483-2488.
29. Michelson AD, Barnard MR, Krueger LA, et al. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin:studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation.2001,104:1533-1537.
30. Ott I, Neumann FJ, Gawaz M, et al. Increased neutrophil-platelet adhesion in patients with unstable angina. Circulation.1996,94:1239-1246.
31. Sarma J, Laan CA, Alam S, et al. Increased platelet binding to circulating monocytes in acute coronary syndromes. Circulation.2002,105:2166-2171.
32. Furman MI, Barnard MR, Krueger LA, et al. Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction. J Am Coll Cardiol. 2001,38:1002-1006.
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Management of Patients with Chronic Stable Angina). J Am Coll Cardiol,1999,33 (7):2092-2197.
2. Braunwald E, Antman EM, Beasley JW, et al. ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocardial infarction-summary article:a report of the American College of Cardiology/American Heart Association task force on practice guidelines (Committee on the Management of Patients with Unstable Angina). J Am Coll Cardiol,2002,40 (7):1366-1374.
3. Lundahl J, Jacobson SH, Paulsson JM. IL-8 from local subcutaneous wounds regulates CD11b activation. Scand J Immunol.2012,75(4):419-425.
4. Li N, Goodall AH, Hjemdahl P. Efficient flow cytometric assay for platelet-leukocyte aggregates in whole blood using fluorescence signal triggering. Cytometry. 1999,35(2):154-161.
5. Moriconi A, Cesta MC, Cervellera MN. Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2. J Med Chem.2007,50(17):3984-4002.
6. Takami M, Terry V, Petruzzelli L. Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1. J Immunol.2002; 168(9):4559-4566.
7. Remick GD. Interleukin-8. Crit Care Med.2005,33:s646-s647.
8. Apostolopoulos J, Davenport P, Tipping PG Interleukin-8 production by macrophages from atheromatous plaques. Arterioscler Thromb VascBiol.1996,16: 1007-1012.
9. Geisel J, Jodden V, Obeid R, et al. Stimulatory effect of homocysteine on interleukin-8 expression in human endothelial cells. Clin Chem Lab Med.2003, 41(8):1045-1048.
10. Dje N Guessan P, Riediger F, Vardarova K, et al. Statins control oxidized LDL-mediated histone modifications and gene expression in cultured human endothelial cells. Arterioscler Thromb Vasc Biol.2009,29:380-386.
11. Ryoo SW, Kim DU, Won M, et al. Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovasc Res.2004,62(1):185-193.
12. Inoue T, Hiroshi Komoda, Masako Nonaka, et al. Interleukin-8 as an independent predictor of long-term clinical outcome in patients with coronary artery disease. International Journal of Cardiology.2008,124(3):319-325.
13. Herder C,Baumert J,Thorand B,et al. Chemokines and incident coronary heart disease:results from the MONICA/KORA Augsburg case-cohort study,1984-2002. Arterioscler Thromb Vasc Biol.2006,9:2147-2152
14. Gerszten RE, Garcia-Zepeda EA, Lim YC, et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature.1999, 398:718-723.
15. Koch AE, Volin MV, Woods JM, et al. Regulation of angiogenesis by the C-X-C chemokines interleukin-8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint. Arthritis Rheum.2001,44(1):31-40.
16. Strieter RM, Kunkel SL, Elner VM, et al. Interleukin-8. A corneal factor that induces neovascularization. Am J Pathol.1992,141(6):1279-1284.
17.殷惠军,马晓娟,蒋跃绒,等.冠心病差异基因表达谱的构建及目标基因的功能分析.科学通报,2009,54(3)：354-359.
18. Chen VM, Hoqq PT. Auosteric disulfide in thrombosis and thrombolysis. J thromb Haemost.2006,4:2533-2541.
19. Rauch U, Nemerson Y. Tissue factor, the blood, and the arterial wall. Trends Cardiovasc Med.2000;10(4):139-143.
20. Luther T, Mackman N. Tissue factor in the heart:Multiple roles in hemostasis, thrombosis, and inflammation. Trends Cardiovasc Med.2001,11:307-312.
21. Zilmann A, Luther T, Kotzsch M, et al.Patelet associated tissue factor contributes to the collagen triggered activation of blood coagulation. Biophs ReS Commun.2001, 281 (2):603-609.
22. Plescia J, Altieri DC. Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leukocyte initiation of coagulation. Biochem J.1996,319(3):873-879.
1. Moriconi A, Cesta MC, Cervellera MN. Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2. J Med Chem.2007,50(17):3984-4002.
2. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines±CXC and CC chemokines. Adv Immunol.1994,55:97-179.
3. Baggiolini M, Dewald B, Moser B. Human chemokines:an update. Annu Rev Immunol.1997,15:675-705.
4. Moore BB, Keane MP, Addison CL, et al. CXC chemokine modulation of angiogenesis:the importance of balance between angiogenic and angiostatic members of the family. J Investig Med.1998,46:113-120.
5. Gear AR, Camerini D.Platelet chemokines and chemokine receptors:linking hemostasis, inflammation, and host defense. Microcirculation.2003,10(3-4): 335-350.
6. Aukrust P, Halvorsen B, Yndestad A, et al. Chemokines and cardiovascular risk. Arterioscler Thromb Vasc Biol.2008,28(11):1909-1919.
7. Fredriksson, R., M. C. Lagerstrom, L. G Lundin, et al. The G-protein-coupled receptors in the human genome form five main families:phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol.2003,63:1256-1272.
8. Rose Marie Stillie, Shukkur Muhammed Farooq, John R. Gordon et al.The functional significance behind expressing two IL-8 receptor types on PMN.Journal of Leukocyte Biology.2009,86:529-543.
9. Gimbrone MA Jr, Obin MS, Brock AF, et al. Endothelial interleukin-8:a novel inhibitor of leukocyte-endothelial interactions. Science.1989,246:1601-1603.
10. Thomas, K. M.Taylor, L.Navarro. The interleukin-8 receptor is encoded by a neutrophil-specific cDNA clone, F3R. J. Biol. Chem.1991,266:14839-14841.
11. Takata, H.,Tomiyama, H.Fujiwara, et al. Cutting edge:expression of chemokine receptor CXCRl on human effector CD8 T cells. J. Immunol.2004,173:2231-2235.
12. Lippert, U.Zachmann, K.Henz, et al. Human T lymphocytes and mast cells differentially express and regulate extra-and intracellular CXCR1 and CXCR2. Exp. Dermatol.2004,13:520-525.
13. Power CA, Clemetson JM, Clemetson KJ, et al. Chemokine and chemokine receptor mRNA expression in human platelets.Cytokine.1995,(7)6:479-482.
14. Clemetson KJ, Clemetson JM, Proudfoot AE, et al.Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets. Blood. 2000,(96)13:4046-4054.
15.李家增,贺石林,王鸿利.血栓病学.北京：科学出版社,1998.P166.
16. Dajani EZ, Shahwan TG, Dajani NE. Statins,platelet aggregation and coronary heart disease. J Assoc Acad Minor Phys.2002,13(1):27-31.
1. Lundahl J, Jacobson SH, Paulsson JM. IL-8 from local subcutaneous wounds regulates CD11b activation. Scand J Immunol.2012,75(4):419-425.
2. Moriconi A, Cesta MC, Cervellera MN. Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2. J Med Chem.2007,50(17):3984-4002.
3. Takami M, Terry V, Petruzzelli L. Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1. J Immunol.2002; 168(9):4559-4566.
4. Reynaudaf Geijersstam A, Sorsa T, Stackelberg S. Effect of E. faecalis on the release of serine proteases elastase and cathepsin G, and collagenase-2 (MMP-8) by human polymorphonuclear leukocytes (PMNs). Int Endod J,2005,38(9):667-767.
5. Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89:3503-3521.
6. Sommerhoff CP, Nadel JA, Basbaum CB, Caughey GH.Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest 1990;85:682-689.
7. Hanson RD, Connolly NL, Burnett D, et al. Developmental regulation of the human cathepsin G gene in myelomonocytic cells. J Biol Chem.1990,265: 1524-1530.
8. Owen JS, Baker PR, O'Flaherty JT. Stress-induced platelet-activating factor synthesis in human neutrophils. Biochim Biophys Acta.2005,1733:120-129.
9. Aukrust P, Halvorsen B, Yndestad A, et al. Chemokines and cardiovascular risk. Arterioscler Thromb Vasc Biol.2008,28(11):1909-1919.
10. Saeed SA, Rasheed H. Calcium-dependent synergistic interaction of platelet activating factor and epinephrine in human platelet aggregation. Acta Pharmacol Sin, 2003,24(1):31-36.
11. Cerletti C, Evangelista V, Molino M. Platelet activation by polymorphonuclear leukocytes:role of cathepsin G and P-selectin. Thromb Haemost.1995,74(1): 218-223.
12. Sambrano GR, Huang W, Faruqi T, et al. Cathepsin G activates protease-activated receptor-4 in human platelets. J Biol Chem.2000,275(10): 6819-6923.
13. Weber C. Platelets and chemokines in atherosclerosis:partners in crime. Circ Res.2005,96(6):612-616.
14. Gale AJ, Rozenshteyn D. Cathepsin G, a leukocyte protease, activates coagulation factor VIII. Thromb Haemost.2008,99(1):44-51.
2. Tyers, M.,& Mann,M. From genomics to proteomics.Nature,2003,422:193-197.
3. Mahesh Uttamchandani, Junqi Li, Hongyan Sun, et al. Yao. Activity-Based Protein Profiling:New Developments and Directions in Functional Proteomics. ChemBioChem, 2008,9:667-675.
4. Thomas Kocher, Giulio Superti-Furga.Mass spectrometry-based functional proteomics: from molecular machines to protein networks. Nature methods,2007,4(10):807-815.
5.杨保林,王阶,姜燕.反向Northern筛查冠心病血瘀证差异片段.中国中医基础医学杂志,2004,10(9)：669-674.
6.马晓娟,殷惠军,陈可冀.血瘀证患者差异基因表达谱研究.中西医结合报,2004,6(4)：355-360.
7.赵慧辉,王伟,郭淑贞.冠心病不稳定型心绞痛血瘀证的蛋白质组学.中国动脉硬化杂志,2008,16(7)：545-548.
8.吴红金,马增春,高月,等.蛋白质组学技术对冠心病血瘀证相关蛋白的研究.中西医结合心脑血管病杂志2005,3(3)189-191.
9.周倩倩,李治国,黄力.冠心病血瘀证的血清差异蛋白质组学研究.微循环学杂志,2012,22(1)：13-16.
10.刘剑刚,徐浩,董国菊,等.血府逐瘀口服液对冠心病心绞痛患者血小板活化分子表达的影响.长春中医药大学学报,2007,23：29-31.
11. Chen KJ, Xue M, Yin H J. The relationship between platelet activation and coronary heart disease and blood stasis syndrome. Shou Du Yi Ke Da Xue Xue Bao.2008,29(3): 266-269.
12.吴锦,陈可冀,裴正礼,等.冠心病血瘀证患者血小板扫描电镜形态观察.中国医药学报,1990,5(1)：18-20.
13.杨军辉,周小青.冠心病心绞痛血瘀证四亚型与血小板活性相关性探讨.中国中医药咨讯,2011,3(7)：306-307.
14.李雪峰,蒋跃绒,高铸烨,等.冠心病血瘀证血小板差异功能蛋白筛选、鉴定及功能分析.中国中西医结合杂志,2010,30(5)：467-473.
15.殷惠军,马晓娟,蒋跃绒,等.冠心病差异基因表达谱的构建及目标基因的功能分析.科学通报,2009,54(3)：354-359.
16. Ye Huang, Huijun Yin, Jingshang Wang, et al. Aberrant expression of Fc γRIIIA (CD16) contributes to the development of atherosclerosis. Gene.2012,498(l):91-95.
17. LIU Yue, YIN Huijun, JIANG Yuerong, et al. Correlation between platelet gelsolin level and different types of coronary heart disease. Chin Sci Bull,2012,57(6):631-638.
1. Remick GD. Interleukin-8. Crit Care Med,2005,33:s646-s647.
2. Waugh JJD, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res,2008, 14:6735-6741.
3. Standiford TJ, Kunkel SL, Basha MA, et al. Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung. J Clin Invest,1990,86:1945-1953.
4. Gimbrone MA Jr, Obin MS, Brock AF, et al. Endothelial interleukin-8:a novel inhibitor of leukocyte-endothelial interactions. Science,1989,246:1601-1603.
5. RoseMarie Stillie, Shukkur Muhammed Farooq, John R. Gordon, et al.The functional significance behind expressing two IL-8 receptor types on PMN.Journal of Leukocyte Biology,2009,86:529-543.
6. DeForge LE, Fantone JC, Kenney JS, et al. Oxygen radical scavengers selectively inhibit interleukin-8 production in human whole blood. J Clin Invest,1992,90: 2123-2129.
7. Rus HG, Vlaicu R, Niculescu F. Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall. Atherosclerosis,1996,127:263-271.
8. Apostolopoulos J, Davenport P, Tipping PG. Interleukin-8 production by macrophages from atheromatous plaques. Arterioscler Thromb Vase Biol,1996, 16:1007-1012.
9. Geisel J, Jodden V, Obeid R, et al. Stimulatory effect of homocysteine on interleukin-8 expression in human endothelial cells. Clin Chem Lab Med,2003, 41(8):1045-1048.
10. Dje N Guessan P, Riediger F, Vardarova K, et al. Statins control oxidized LDL-mediated histone modifications and gene expression in cultured human endothelial cells. Arterioscler Thromb Vasc Biol,2009,29:380-386.
11. Ryoo SW, Kim DU, Won M, et al. Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovasc Res,2004,62 (1):185-193.
12. Wang YS, Li XJ, Zhao WO. TREM-1 is a positive regulator of TNF-a and IL-8 production in U937 foam cells. Bosn J Basic Med Sci,2012,12(2):94-101.
13. Al-Bannawi A, Al-Wesebai K, Taha S, et al. Chlamydia pneumoniae induces chemokine expression by platelets in patients with atherosclerosis. Med Princ Pract, 2011,20(5):438-443.
14. Deng S, Jepsen S, Dommisch H, et al. Cysteine proteases from Porphyromonas gingivalis and TLR ligands synergistically induce the synthesis of the cytokine IL-8 in human artery endothelial cells. Arch Oral Bio,2011,56(12):1583-1591.
15. Gerszten RE, Garcia-Zepeda EA, Lim YC, et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature,1999, 398:718-723.
16. Koch AE, Volin MV, Woods JM, et al. Regulation of angiogenesis by the C-X-C chemokines interleukin-8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint. Arthritis Rheum,2001,44(1):31-40.
17. Strieter RM, Kunkel SL, Elner VM, et al. Interleukin-8. A corneal factor that induces neovascularization. Am J Pathol,1992,141(6):1279-1284.
18. Arenberg DA, Kunkel SL, Polverini PJ, et al. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J Clin Invest,1996, 12:2792-2802.
19. Simonini A, Moscucci M, Muller DW, et al. IL-8 is an angiogenic factor in human coronary atherectomy tissue. Circulation,2000,101:1519-1526.
20. Li A, Dubey S, Varney ML, et al. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol,2003,170(6):3369-3376.
21. Kyriakakis E, Cavallari M, Pfaff D, et al. IL-8-mediated angiogenic responses of endothelial cells to lipid antigen activation of iNKT cells depend on EGFR transactivation. J Leukoc Biol,2011,90(5):929-939.
22. Luca M, Huang S, Gershenwald JE, et al. Expression of interieukin-8 by human melanoma cells up-regulates MMP-2 activity and increases tumor growth and metastasis. Am J Pathol,1997,151:1105-1113.
23. Moreau M, Brocheriou I, Petit L, et al. Interleukin-8 mediates downregulation of tissue inhibitor of metalloproteinase-1 expression in cholesterol-loaded human macrophages:relevance to stability of atherosclerotic plaque. Circulation,1999, 99:420-426.
24. Teruo Inoue, Hiroshi Komoda, Masako Nonaka, et al. Interleukin-8 as an independent predictor of long-term clinical outcome in patients with coronary artery disease. International Journal of Cardiology,2008,124(3):319-325.
25. Herder C,Baumert J,Thorand B,et al. Chemokines and incident coronary heart disease:results from the MONICA/KORA Augsburg case-cohort study,1984-2002. Arterioscler Thromb Vasc Biol,2006,9:2147-2152.
26. Elmas E, Lang S, Dempfle CE, et al. High plasma levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and interleukin-8 (IL-8) characterize patients prone to ventricular fibrillation complicating myocardial infarction. Clin Chem Lab Med,2007, 45(10):1360-1365.
27. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez M, et al. Prognostic value of interleukin-8 as a predictor of heart failure in patients with myocardial infarction and percutaneous intervention. International Journal of Cardiology,2006,111(1):158-160.
28. De Gennaro L, Brunetti ND, Montrone D, et al. Subacute inflammatory activation in subjects with acute coronary syndrome and left ventricular dysfunction. Inflammation,2012,35(1):363-370.
29. Damas JK, Eiken HG, Oie E, et al. Myocardial expression of CC-and CXC-chemokines and their receptors in human end-stage heart failure. Cardiovasc Res,2000,47(4):778-787.
30. Leonard DA, Merhige ME, Williams BA, et al. Elevated expression of the interleukin-8 receptors CXCR1 and CXCR2 in peripheral blood cells in obstructive coronary artery disease. Coron Artery Dis,2011,22(7):49-49
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Management of Patients with Chronic Stable Angina). J Am Coll Cardiol.1999,33 (7):2092-2197.
2. Braunwald E, Antman EM, Beasley JW, et al. ACC/AHA 2002 guideline updatefor the management of patients with unstable angina and non-ST-segment elevation myocardial infarction-summary article:a report of the American College of Cardiology/American Heart Association task force on practice guidelines (Committee on the Management of Patients with Unstable Angina). J Am Coll Cardiol.2002,40 (7):1366-1374.
3. 卫生部心血管防治研究中心.中国心血管病报告2011.北京：中国大百科全书出版社,2012.
4. Chierchia SL. Inflammation and acute coronary syndromes. Rev Esp Cardiol, 2001,54(10):1135-1140.
5. Li N, Hu H, Lindqvist M, et al. Platelet-leukocyte cross talk in whole blood. Arterioscler Thromb Vasc Biol.2000,20(12):2702-2708.
6. Ruggeri ZM. Platelets in atherothrombosis. Nat Med.2002,8:1227-1234.
7. Gawaz M. Do platelets trigger atherosclerosis? Thromb Haemost.2003, 90:971-972.
8. 李家增,贺石林,王鸿利.血栓病学.北京：科学出版社,1998.P25-27.
9. Picker SM. Platelet function in ischemic heart disease. J Cardiovasc Pharmacol. 2013,61(2):166-174.
10. Weber C. Platelets and chemokines in atherosclerosis:partners in crime. Circ Res.2005,96(6):612-616.
11. Ross R. Atherosclerosis-an inflammatory disease. N Eng J Med.1999, 340:115-126.
12. Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation.2001,104:365-372.
13. Biasucci LM, D Onofrio G, Liuzzo G, et al. Intracellular neutrophil myeloperoxidase is reduced in unstable angina and myocardial infarction, but its reduction is not related to ischemia. J Am Coll Cardiol.1996,27:611-616.
14. Home BD, Anderson JL, John JM et al. Intermountain Heart Collaborative Study Group. Which white blood cell subtypes predict increased cardiovascular risk?. J Am Coll Cardiol.2005,45:1638-1643.
15. Gillum RF, Mussolino ME, Madans JH. Counts of Neutrophils, Lymphocytes, and Monocytes, Cause-specific Mortality and Coronary Heart Disease:The NHANES-I Epidemiologic Follow-up Study. Ann Epidemiol.2005,15:266-271.
16. Bharadwaj D, Stein MP, Volzer M, et al.The major recept or for C- reactive protein on leukocytes is fcgamma receptor. J Exp Med.1999,190(4):585-590.
17. Ott I, Neumann FJ, Kenngott S, et al. Procoagulant inflammatory responses of monocytes after direct balloon angioplasty in acute myocardial infarction. Am J Cardiol.1998,82:938-942.
18. Engler RL, Schmid-Schonbein GW, Pavelec RS. Leokocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol.1983,111:98-111.
19. Zatta A, Prosdocimi M, Bertere V, et al. Inhibition of platelet function by polymorphonuclear leukocytes. J Lab Clin Med.1990,116:651-660.
20. Pidard D, Renesto P, Rahbi S, et al. Regulation of the structure and activity of platelet adhesion receptors by leukocyte proteinases. Nouv Rev Fr Hematol.1994, 36:S99-101.
21. Neuman FJ, Ott I, Marx N, et al. Effect of human recombinant interleukin-6 and interleukin-8 on monocyte procoagulant activity. Arterioscler Thromb Vasc Biol. 1997,17:3399-3405.
22.陈可冀.活血化瘀研究与临床[M]北京：中国协和医科大学,北京医科大学联合出版社,1993,13-17.
23.翁维良.冠心病血瘀证的研究进展.中医杂志.1987,6：464.
24. Habazettl H, Hanusch P, Kupatt C. Effects of endothelium leukocytes platelet interaction on myocardial ischemia-reperfusion injury. Z Kardiol.2000,89 (Supp 19):Ⅸ92-95.
25. Wang J, Zhang S, Jin Y, et al. Elevated levels of platelet monocyte aggregates and related circulating biomarkers in patients with acute coronary syndrome.Int J Cardiol.2007;115(3):361-365.
26. Field EJ, MacLeod I. Platelet adherence to neutrophils. Br Med J.1963, 2:388-389.
27. Konstantopoulos K, Neelamegham S, Burns AR, et al. Venous levels of shear support neutrophil platelet adhesion and neutrophil aggregation in blood via P-selectin and β2-integrin. Circulation.1998,98:873-882.
28. E ppihim er M J, S chaub RG. P-S elect in-dependent inhibit ion of thrombosis during venous stasis. Arterioscler Thromb Vasc B io.2000,20:2483-2488.
29. Michelson AD, Barnard MR, Krueger LA, et al. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin:studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation.2001,104:1533-1537.
30. Ott I, Neumann FJ, Gawaz M, et al. Increased neutrophil-platelet adhesion in patients with unstable angina. Circulation.1996,94:1239-1246.
31. Sarma J, Laan CA, Alam S, et al. Increased platelet binding to circulating monocytes in acute coronary syndromes. Circulation.2002,105:2166-2171.
32. Furman MI, Barnard MR, Krueger LA, et al. Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction. J Am Coll Cardiol. 2001,38:1002-1006.
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Management of Patients with Chronic Stable Angina). J Am Coll Cardiol,1999,33 (7):2092-2197.
2. Braunwald E, Antman EM, Beasley JW, et al. ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocardial infarction-summary article:a report of the American College of Cardiology/American Heart Association task force on practice guidelines (Committee on the Management of Patients with Unstable Angina). J Am Coll Cardiol,2002,40 (7):1366-1374.
3. Lundahl J, Jacobson SH, Paulsson JM. IL-8 from local subcutaneous wounds regulates CD11b activation. Scand J Immunol.2012,75(4):419-425.
4. Li N, Goodall AH, Hjemdahl P. Efficient flow cytometric assay for platelet-leukocyte aggregates in whole blood using fluorescence signal triggering. Cytometry. 1999,35(2):154-161.
5. Moriconi A, Cesta MC, Cervellera MN. Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2. J Med Chem.2007,50(17):3984-4002.
6. Takami M, Terry V, Petruzzelli L. Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1. J Immunol.2002; 168(9):4559-4566.
7. Remick GD. Interleukin-8. Crit Care Med.2005,33:s646-s647.
8. Apostolopoulos J, Davenport P, Tipping PG Interleukin-8 production by macrophages from atheromatous plaques. Arterioscler Thromb VascBiol.1996,16: 1007-1012.
9. Geisel J, Jodden V, Obeid R, et al. Stimulatory effect of homocysteine on interleukin-8 expression in human endothelial cells. Clin Chem Lab Med.2003, 41(8):1045-1048.
10. Dje N Guessan P, Riediger F, Vardarova K, et al. Statins control oxidized LDL-mediated histone modifications and gene expression in cultured human endothelial cells. Arterioscler Thromb Vasc Biol.2009,29:380-386.
11. Ryoo SW, Kim DU, Won M, et al. Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovasc Res.2004,62(1):185-193.
12. Inoue T, Hiroshi Komoda, Masako Nonaka, et al. Interleukin-8 as an independent predictor of long-term clinical outcome in patients with coronary artery disease. International Journal of Cardiology.2008,124(3):319-325.
13. Herder C,Baumert J,Thorand B,et al. Chemokines and incident coronary heart disease:results from the MONICA/KORA Augsburg case-cohort study,1984-2002. Arterioscler Thromb Vasc Biol.2006,9:2147-2152
14. Gerszten RE, Garcia-Zepeda EA, Lim YC, et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature.1999, 398:718-723.
15. Koch AE, Volin MV, Woods JM, et al. Regulation of angiogenesis by the C-X-C chemokines interleukin-8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint. Arthritis Rheum.2001,44(1):31-40.
16. Strieter RM, Kunkel SL, Elner VM, et al. Interleukin-8. A corneal factor that induces neovascularization. Am J Pathol.1992,141(6):1279-1284.
17.殷惠军,马晓娟,蒋跃绒,等.冠心病差异基因表达谱的构建及目标基因的功能分析.科学通报,2009,54(3)：354-359.
18. Chen VM, Hoqq PT. Auosteric disulfide in thrombosis and thrombolysis. J thromb Haemost.2006,4:2533-2541.
19. Rauch U, Nemerson Y. Tissue factor, the blood, and the arterial wall. Trends Cardiovasc Med.2000;10(4):139-143.
20. Luther T, Mackman N. Tissue factor in the heart:Multiple roles in hemostasis, thrombosis, and inflammation. Trends Cardiovasc Med.2001,11:307-312.
21. Zilmann A, Luther T, Kotzsch M, et al.Patelet associated tissue factor contributes to the collagen triggered activation of blood coagulation. Biophs ReS Commun.2001, 281 (2):603-609.
22. Plescia J, Altieri DC. Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leukocyte initiation of coagulation. Biochem J.1996,319(3):873-879.
1. Moriconi A, Cesta MC, Cervellera MN. Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2. J Med Chem.2007,50(17):3984-4002.
2. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines±CXC and CC chemokines. Adv Immunol.1994,55:97-179.
3. Baggiolini M, Dewald B, Moser B. Human chemokines:an update. Annu Rev Immunol.1997,15:675-705.
4. Moore BB, Keane MP, Addison CL, et al. CXC chemokine modulation of angiogenesis:the importance of balance between angiogenic and angiostatic members of the family. J Investig Med.1998,46:113-120.
5. Gear AR, Camerini D.Platelet chemokines and chemokine receptors:linking hemostasis, inflammation, and host defense. Microcirculation.2003,10(3-4): 335-350.
6. Aukrust P, Halvorsen B, Yndestad A, et al. Chemokines and cardiovascular risk. Arterioscler Thromb Vasc Biol.2008,28(11):1909-1919.
7. Fredriksson, R., M. C. Lagerstrom, L. G Lundin, et al. The G-protein-coupled receptors in the human genome form five main families:phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol.2003,63:1256-1272.
8. Rose Marie Stillie, Shukkur Muhammed Farooq, John R. Gordon et al.The functional significance behind expressing two IL-8 receptor types on PMN.Journal of Leukocyte Biology.2009,86:529-543.
9. Gimbrone MA Jr, Obin MS, Brock AF, et al. Endothelial interleukin-8:a novel inhibitor of leukocyte-endothelial interactions. Science.1989,246:1601-1603.
10. Thomas, K. M.Taylor, L.Navarro. The interleukin-8 receptor is encoded by a neutrophil-specific cDNA clone, F3R. J. Biol. Chem.1991,266:14839-14841.
11. Takata, H.,Tomiyama, H.Fujiwara, et al. Cutting edge:expression of chemokine receptor CXCRl on human effector CD8 T cells. J. Immunol.2004,173:2231-2235.
12. Lippert, U.Zachmann, K.Henz, et al. Human T lymphocytes and mast cells differentially express and regulate extra-and intracellular CXCR1 and CXCR2. Exp. Dermatol.2004,13:520-525.
13. Power CA, Clemetson JM, Clemetson KJ, et al. Chemokine and chemokine receptor mRNA expression in human platelets.Cytokine.1995,(7)6:479-482.
14. Clemetson KJ, Clemetson JM, Proudfoot AE, et al.Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets. Blood. 2000,(96)13:4046-4054.
15.李家增,贺石林,王鸿利.血栓病学.北京：科学出版社,1998.P166.
16. Dajani EZ, Shahwan TG, Dajani NE. Statins,platelet aggregation and coronary heart disease. J Assoc Acad Minor Phys.2002,13(1):27-31.
1. Lundahl J, Jacobson SH, Paulsson JM. IL-8 from local subcutaneous wounds regulates CD11b activation. Scand J Immunol.2012,75(4):419-425.
2. Moriconi A, Cesta MC, Cervellera MN. Design of noncompetitive interleukin-8 inhibitors acting on CXCR1 and CXCR2. J Med Chem.2007,50(17):3984-4002.
3. Takami M, Terry V, Petruzzelli L. Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1. J Immunol.2002; 168(9):4559-4566.
4. Reynaudaf Geijersstam A, Sorsa T, Stackelberg S. Effect of E. faecalis on the release of serine proteases elastase and cathepsin G, and collagenase-2 (MMP-8) by human polymorphonuclear leukocytes (PMNs). Int Endod J,2005,38(9):667-767.
5. Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997; 89:3503-3521.
6. Sommerhoff CP, Nadel JA, Basbaum CB, Caughey GH.Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest 1990;85:682-689.
7. Hanson RD, Connolly NL, Burnett D, et al. Developmental regulation of the human cathepsin G gene in myelomonocytic cells. J Biol Chem.1990,265: 1524-1530.
8. Owen JS, Baker PR, O'Flaherty JT. Stress-induced platelet-activating factor synthesis in human neutrophils. Biochim Biophys Acta.2005,1733:120-129.
9. Aukrust P, Halvorsen B, Yndestad A, et al. Chemokines and cardiovascular risk. Arterioscler Thromb Vasc Biol.2008,28(11):1909-1919.
10. Saeed SA, Rasheed H. Calcium-dependent synergistic interaction of platelet activating factor and epinephrine in human platelet aggregation. Acta Pharmacol Sin, 2003,24(1):31-36.
11. Cerletti C, Evangelista V, Molino M. Platelet activation by polymorphonuclear leukocytes:role of cathepsin G and P-selectin. Thromb Haemost.1995,74(1): 218-223.
12. Sambrano GR, Huang W, Faruqi T, et al. Cathepsin G activates protease-activated receptor-4 in human platelets. J Biol Chem.2000,275(10): 6819-6923.
13. Weber C. Platelets and chemokines in atherosclerosis:partners in crime. Circ Res.2005,96(6):612-616.
14. Gale AJ, Rozenshteyn D. Cathepsin G, a leukocyte protease, activates coagulation factor VIII. Thromb Haemost.2008,99(1):44-51.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |