| Background&Objective:Coronary heart disease (CHD) has become the primary threat to human life diseases, and acute myocardial infarction (AMI) are the most serious one in different stages of development of coronary heart disease, its main pathology foundation is atherosclerosis (AS) formation. Study shows that:occurrence and development of AMI are extremely complex process, involving endothelial cell injury, inflammatory cell infiltration, plaque and thrombosis, crack, etc.At present, percutaneous transluminal coronary angioplasty (percu-taneous coronary intervention, PCI) has become one of the most effective the treatment of CHD. Although it have made some effect to thrombolytic therapy with some drugs in prevention of rethrombosis and the prevention of reinfarction after the vascular recanalization, but because of the vascular endothelial damagment and stimulating inflammatory response on surgery, and there will be vascular endothelial damagment and thrombus formation in some postoperative patients, so the late impact of PTCA is seriously effected. The injury of vascular endothelial cell is the first and core injury in PCI and the important causes of restenosis. It is necessary of re-endothelialization of damaged blood vessels and restore endothelial function in the maintenance of vascular homeostasis within the environment, inhibiting inflammatory reaction and abnormal hyperplasia of vascular smooth muscle as soon as possible, that is an effective means to to prevent restenosis after PCI. Vascular endothelial injury and dysfunction in many diseases are initiated, through the whole process of disease. Its cause vascular endothelial dysfunction, and hypertension, atherosclerosis, heart failure, diabetes and other cardiovascular and cerebrovascular diseases and metabolic diseases are closely related to the occurrence and development. Recent studies have found that endothelial progenitor cells (EPCs) with specific homing in hypoxia-ischemia, vascular injury, and promote the characteristics of the formation of new blood vessels, so when the body itself, excessive consumption of EPCs due to insufficient, external EPCs transplantation in the treatment of vascular endothelial injury disease has become a new research focus.Research has found that inflammatory response are important characteristics of AS plaques and have a certain degree of associated on macrophages, endothelial cells, smooth muscle cells (SMC) activation and proliferation, cytokine and growth factor production, complement activation and unperturbed, as well as the other emergence of inflammatory mediators.The inflammatory response is of great significance in the occurrence and development of AS. To intimal injury, the formation of AS is not only a process of immune response, but also a process of inflammatory response. Among the various studies about inflammatory factors, the IL-18 is a recent hotspot. IL-18 is a major cell-mediated immune pleiotropic cytokine, and its structure similar to IL-1 receptor, functions similar to IL-12, with a wide range of biological activity. It mainly induces to product interferon-γ(IFN-γ),and plays a synergistic role with cooperation of IL-12, and enhances lysis activity of the natural killer cells and tumor-specific anti-(T lymphoid) cells, and plays an important role at AMI and I/R inflammatory response. The study found that there is a high sensitivity to IL-18 in cardiomyocytes. IL-18 may diminish cardiac function and induce myocardial injury, but the changes of IL-18 is not yet fully understand at the patients of AMI. There is little study about the chemical inflammatory response and its detailed change at home and abroad when AMI occurs, and it is rarely reported to the condition of dynamic change and the relevance of studies of IL-18 in AMI patients.Recent studies have found that endothelial progenitor cells (EPCs)have the characteristics with specific homing to ischemia and hypoxia, vascular damage tissue, and promoting the formation of new blood vessels.Transplanted EPCs promote angiogenesis mechanism may have two aspects:One is migrated to the location into mature vascular endothelial cells; Second, secretion of angiopoietin substances through autocrine and paracrine mechanism, such as VEGF, HGF (hepatocyte growth factor), IGF-1 (insulin-like growth factor 1), G-CSF, etc; EPCs can differentiate into myocardial cells horizontally. In recent years, the applications of EPCs transplantation in cardiovascular disease were mainly to lower extremity ischemia, ischemic myocardium and promote angiogenesis therapy research, as well as vascular injury, artificial blood vessels and stent endothelialization. Experiments show that EPCs transplantation can significantly improve blood flow in ischemic area, increased capillary density, and promote functional recovery and play an active role in damaged blood vessels, artificial blood vessels and endothelial stent to prevent restenosis.The coronary thrombosis is the most common cause in AMI with ST egment (AMI) accounting for more than 80-90% of all cause of. Clinically, It has been proved effective by intravenous fibrinolytic therapy. In recent years, it shows that snake venom fibrinolytic enzyme has growth factor activity and nutrition effects on the cells. It was good to inject EPCs and fibrinolytic enzyme into the rabbit model of myocardial infarction. However, it was not yet clear that how to promote vascular endothelial recovery in thrombolytic therapy for snake venom and how to suppress the inflammatory cytokines on vascular endothelial further injury, and the research is relatively rare in particular venom thrombolysis complemented by autologous progenitor cell transplantation.This study focused on the effection of reperfusion of blood flow in patients with cardiovascular disease and recovery of injuried endothelial, as well as the control of inflammatory cytokines in the inflammatory response by the therapy with venom thrombolytic and transplantation of endothelial progenitor cells derived from the vivo. First of all, the peripheral blood endothelial progenitor cells were separated in the patients with coronary artery disease, and cultured identified. We observe the changes in quantity and function of endothelial progenitor cells,measure the concentration of serum IL-18. Secondly, to explore the therapeutic effect of thrombolytic therapy in snake venom, the number and function of endothelial progenitor cells and concentration of IL-18 were checked. ..Finally we establish animal model of vascular injury, then transplante endothelial progenitor cells derived from the vivo on the basis of the venom thrombolytic therapy. and finally observe the recovery after endothelial injury and the concentration changes of serum IL-18 in inflammatory response, aim to provide new ideas and theoretical basis to cell transplantation in the clinical treatment of cardiovascular diseaseMethod1.Experimental group(1) Case Information:There were 200 cases of hospitalized patients with coronary artery disease at Zhujiang Hospital, Southern Medical University in 2006 October to 2007 October,45 cases with stable angina,40 cases of unstable angina,115 cases with acute myocardial infarction (45 cases of single lesions,38 cases of double vessel disease,32 cases of multivessel disease),100 cases of AMI with venom thrombolytic therapy were divided into before treatment group and after treatment group.,while 20 cases of healthy people as controls.(2) Animal grouping:An average of 28 Japanese rabbits were divided into four groups:A-culture medium control group, B-cell transplantation group, C-venom plasminogen Group, D-venom plasminogen and cell transplantation group2. Specimen collection:(1) Blood:All patients hospitalized were blooded from 20ml with heparin anticoagulation, the same to control group in the morning, Blood was collected for the patients before and after thrombolytic therapy, and for the animals dealed after 28 days,dealing with samples 4 hours later. (2) Pathology specimen collection:experimental animals were killed for pathological slices.3. Experimental method(1) The blood EPCs were separated from all coronary heart disease patient group and the venom thrombolytic therapy for culture, identification, counting and functional testing. By density gradient centrifugation and mononuclear cells were isolated, and cultured in the RMP-1640 By DiL-acLDL and FITC-UEA-I double staining, Double-stained cells are considered to be differentiating EPCs.in fluorescence microscope.The cells were counted in the confocal microscope (Count 15 randomly selected field of vision of the EPC) in 200 times. The adherent cells were counted in special fibronectin plate,and their migration cells were counted in Boyden chamber.(2) The concentration of IL-18:Measured the concentration of IL-18, using double-antibody sandwich-enzyme-linked immunosorbent assay (ABC-ELISA)(3) Left anterior descending coronary artery surgery was bar after anesthesia, reperfused an hour later, ECG were carried out in the next bar-end and after reperfusion, and separately injected DMEM culture medium, DMEM culture medium containing endothelial progenitor cells, DMEM culture medium containing snake venom plasminogen, DMEM culture medium containing endothelial progenitor cells and snake venom fibrinolytic enzyme into the artery root of A, B, C, D groups of animals.All animals were first checked by heart ultrasound after transplant surgery 4 weeks,later, and then collected blood, for endothelial progenitor cell count and IL-18 concentration detection.Finally all the animals were sacrificed for biopsy.HE stain and factor VIII immunohistochemistry.10 optional vision calculated as the average number of capillaries stained for the number of new vessels.Results1. Changes of morphology and the number, identification of fluorescent double staining Through density gradient centrifugation, about 3×107 MNC will be collected in 30mL peripheral blood. Cultured 5-6 days later, adherent cells can be seen together to form cell clusters of varying sizes. After cultured 7 days, cell morphology was different from the spindle, round and irregular-shaped.After cultured 7-9 days, a small number of spindle can be seen in small groups late EPC arise at an early stage between the EPC. And in the development of the first 9-12 days, colonies cells as cobblestone were rapidly grown up.It revealed that cell morphology observed under light microscope appeared to be spindle cell and connected into a cord-like, larger and more round in the control group than the treatment group.The treatment group compared with the control group cells had significant differences; the number of cells of AMI group before treatment, was significantly reduction than that of the control group,Mononuclear cells obtained after separated and cultured 7d formed a spindle-like cells of the endothelium. With acLDL-DiL and UEA-I double staining, cells were identificated through confocal microscope, EPCs were charcterized as adherent cells of double positive for acLDL-DiL and UEA-I staining. The number of cells of double-fluorescence staining positive decreased significantly in the patients with AMI of no treatment compared with the control group;2. The number and function of EPCs in coronary heart disease at different stagesIt showed that the numbers of EPCs in stable angina group, unstable angina and myocardial infarction group were significantly lower than that in the control group(P <0.01). The capacity of adhesion (adherent cells) and proliferative of EPCs in unstable angina group decreased significantly than that in stable angina group (P<0. 05); the capacity of adhesion (adherent cells) and proliferative of EPCs in myocardial infarction group also decreased significantly than that in stable angina and unstable angina group (P<0.05).3. The number of EPCs,colony-forming units and concentration of IL-18 in different lesions of AMI before thrombolytic therapy The number of EPCs,colony-forming units in different lesion group of AMI (single, double, multi-vessel disease) were significantly lower than those in the control group (P<0.05); and concentrations of IL-18 were gradually increasing trend, significantly higher than that in the control group (P<0.05); compared many-vessel lesion group with single vessel, double vessel lesion group, the number of EPCs,colony-forming units significantly decreased, IL-18 concentration significantly increased (P<0.05).4. The number of EPCs and concentration of IL-18 before and after thrombolytic therapyThe number of EPCs reduced significantly in AMI group than that in the control group (P<0.01); it increased significantly in the treatment group of treatment 3d than that in no treament group(P<0.05); but compared with the control group there was no obvious difference (P> 0.05); the number of cells after treatment 7d decreased, but still more than that before treatment (P<0.05). The number of colony-forming units (CFU)was less and CFU was smaller and sparser in the AMI patients than those in the control group (P<0.05); The number of CFU increased after treatment 3d,but compared with no treatment there was no significant difference (P>0.05); after cells, there was no difference compared treatment 7d with no treatment, and the number of CFU was less than that in the control group (P<0.05). The concentration of IL-18 in the treatment of three days, seven days was higher than that in the control group(P<0.05), but decreased significantly compared to no treatment (P<0.05).5. Heart functionRabbit coronary artery ligation of left anterior descending coronary artery, the ECG demonstrated ST-segment elevation; one of A group died during surgery. After reperfusion, ST segment declined slowly, five cases appeared tachycardia, two cases appeared bradygastria, D group appeared ventricular fibrillation, one died.Comparison of echocardiography in each group animals, left ventricular ejection fraction (64.9±4.51,66.1±5.46,68.9±5.07), Rate of left ventricular wall motion of B, C, D group (0.26±0.28,0.26±0.18,0.28±0.45)were significantly higher than group A(59.8±2.96,0.22±0.39) (P<0.05); Left ventricular fraction shortening in group B and D (38.1±2.95,39.6±2.31)were significantly higher than group A(34.9±1.61) (P<0.05),while those in group C(35.8±2.64) was near to group A(34.9±1.61) (P=0.16).6.Pathological examinationWhen all experimental animals were killed for myocardial pathology, it showed that myocardial infarction zone in C, A group(1.99±0.47 mm,2.03±0.72mm) was no significant different compared to normal myocardium (1.95±0.89mm); surface color in B, D group myocardial only little change; thickness of B,D ventricular wall myocardium (2.29±0.29mm,2.34±0.17mm) was near to that of normal ventricular wall of the myocardium; thickness of D group (2.34±0.17mm) was more than that of A group (2.03±0.72mm) (P<0.05).For the remaining group, the difference was not obvious. FactorⅧstaining, compared capillary density was found, D group (10.3±3.5) were significantly higher than B, C, A three-group (7.2±2.5,6.9±2.2,3.4±1.7), B, C groups did not differ large, but compared with the A group are also significant differences.7.The number of EPCs and concentration of each group animalsThe number of EPCs and CFU in B, C, D group((39.9±3.9,26.9±5.5; 38.9±5.6, 24.3±4.9; 57.1±12.6,33.8±8.7) increased significantly than that in A group (24.0±1.6,13.8±3.3)(P<0.05);there was a more marked increase compared D group to the B, C group (P<0.05); the concentrations of IL-18 in B, C group (122.5±36.9 pg/ml,119.8±36.5 pg/ml) decreased slightly compared with the A group (142.1±88.5 pg/ml)(P=0.09); but concentrations of IL-18 in D group (40.2±22.1 pg/ml) decreased significantly compared with A group (P<0.05).Conclusions1.Along as different stages of coronary heart disease incidence (stable angina pectoris-unstable angina-acute myocardial infarction) and acute myocardial infarction in different lesions (single-vessel disease-double-vessel disease-multi-vessel disease), vascular injury was increasing and inflammatory response enhanced further, the number and the function of endothelial progenitor cells were decreased.2.The therapy of venom thrombolytic can improve the number of circulating EPCs and inhibited IL-18 expression, thus contributing to the restoration of endothelial injury and reduce the inflammatory response of vascular endothelial further injury.3. The therapy of venom thrombolytic with autologous EPCs transplantation could improve cardiac function necrosis, and it was good to restoration for myocardial infarction.4. The therapy of venom thrombolytic with autologous EPCs transplantation could significantly improve the number of endothelial progenitor cells and colony-and lower the of concentration IL-18, so as to effectively promote the restoration of endothelial injury, control of vascular endothelial damage in inflammatory response.Main innovation:1, It was the first to monitor of peripheral blood progenitor cells in patients with coronary heart disease and the expression of IL-18, and coronary heart disease due to its strong correlation to some extent, so the clinical diagnosis of coronary heart disease can help the sub-type and grade, and its treatment and prognosis is also good clinical significance.2, It was the first animal experiment to show that:the drugs on the basis of thrombolytic therapy combined with autologous progenitor cell transplantation can improve cardiac function, and promote endothelial injury recovery, effectively inhibit the inflammatory response, clinical coronary heart disease, especially acute myocardial infarction provides a new possible treatment. |
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