| BackgroundCoronary atherosclerotic heart disease referred for coronary heart disease (CHD). It is clinically divided into acute coronary syndrome (ACS) and chronic ischemic syndrome. Unstable Angina (UA) is a common type in the ACS. Based on the coronary atherosclerotic lesions, coronary artery intimal hemorrhage occurred and plaque ruptured. Platelets and fibrin aggregated at the breakage site to form thrombosis, leading to acute or subacute myocardial oxygen supply decrease caused by coronary artery spasm and the distal vascular thrombosis. Endothelial progenitor cells (EPCs) is a group of precursor cells existed in the human body which can specifically home in ischemic injury site, and further differentiate into vascular endothelial cells (ECs). Numerous studies have shown that EPCs can promote angiogenesis, repair damage endomembrane, and play an important role in maintaining the steady state of the cardiovascular system. They show a huge potential in the prevention of coronary heart disease.The number of EPCs in peripheral blood of patients with coronary heart disease changes differently in different pathophysiological stages compared to the normal ones. The fact that the number of EPCs in peripheral blood reduces among the chronic ischemic syndrome patients may be related to a number of risk factors for coronary heart disease. On the other hand, the increasing number of EPCs in peripheral blood In patients with acute myocardial infarction may due to the body stimulation by severe myocardial ischemia. According to increasing peripheral blood EPCs, new blood vessels and collateral circulation develop for cardiac perfusion maintaining. However, there was not only because of a number of risk factors for chronic ischemic syndrome patients with unstable angina, but also its unstable coronary plaque easily lead to aggravation of myocardial ischemia. Therefore the problem that the degree of coronary artery disease whether or not related with the amount of peripheral blood EPCs remain controversial in the relevant domestic research or abroad. The treatment of percutaneous coronary intervention (PCI) is an important means for the treatment of coronary artery disease with unstable angina. It can rebuilt stable coronary blood flow, decrease cardiac dysfunction, so as to improve the prognosis and reduce mortality. However, the effect of PCI on the of EPCs number and their biological functions was rarely reported.To explore the relevance between PCI and EPCs, we detected the number of EPCs in the peripheral blood of patients with unstable angina. We gave the coronary disease severity a quantitative score in unstable angina patients through gensini scoring system, then analyzed the correlation between the socre and the indicators, exploring their relevance. On this basis, we gave the unstable angina patients who were consistent with PCI indications the interventional treatment. Then we counted the number of peripheral blood EPCs and double-positive cells, observed cell migration and proliferation ability1week or6months after PCI. The purpose is to further explore the effects of percutaneous coronary intervention therapy on the number of peripheral blood EPCs and their biological function in patients with unstable angina. We hope that we can provide new ideas for the treatment of unstable angina through our research.Purpose1. Through examining the number of peripheral blood EPCs of patients with unstable angina, combined with quantitative ratings of coronary lesions with Gensini score system, we explore the correlation between the lesions severity and the above indicators.2. We explore the impact of percutaneous coronary intervention treatment on the number of EPCs in peripheral blood and double-positive cells of patients with unstable angina, also the impact on cell migration and proliferation capacity of these patients.3. We also explore whether percutaneous coronary intervention for treatment of unstable angina is more conducive to improve the number of peripheral blood EPCs and double positive cells, also the ability of cell migration and proliferation in patients, as compared to pure medication therapy.Methods1. We selected56cases of unstable angina patients who hospitalized in the Department of Cardiology in our hospital from January2011to June2012, ages from45to69years old. They were diagnosed by coronary angiography which is confirmed by the standard made by Chinese Medical Society of Cardiology. All patients were excluded from tumor, severe liver and kidney dysfunction, acute cerebrovascular accident, diabetes and other diseases. Before admission, they were not taking angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB),3-Hydroxy-3-methyl glutarul coenzyme A (HMG-CoA), and reductase inhibitors, etc.2. Coronary artery stenosis assessment All patients are underwent coronary angiography by using Judkins method. We quantitative analyzed coronary artery stenosis according to Gensini scoring system. The scoring method is each segment coronary stenosis score multiplied by each lesion weighting factor, then each part of score composes the final score.3. Postoperative treatment for coronary angiography The patients divided into two groups after angiography.30patients in the intervention group were treated by intervention therapy, planted in Rulei Pa neomycin stent. They got regulatory postoperative treatment by coronary heart disease secondary prevention programs, taking ACEI/ARB drugs, HMG-CoA reductase inhibitors and antiplatelet drugs, etc.26patients in the drug group had standardized treatment by coronary heart disease secondary prevention programs.4. Detection of the number of peripheral blood EPCs and their biological functions Intervention group and drug group completed the following detection in Preoperative,1week and6months Postoperative follow-up respectively.4.1Flow cytometry to detect the number of peripheral blood EPCs We use ethylenediamine tetra-acetic acid (EDTA) tube to extract2ml venous blood, take human peripheral venous blood100μl placed in a flow-type pipe. Then we add phycoerythrin (PE)-labeled mouse anti-human KDR antibodies, Flourescein isothiocyanate (FITC) labeled Ulex lectin1(FITC-UEA-1) labeled mouse anti-human CD34antibody10u1. Negative control tube was added10μl PE, FITC, and allophycocyanin (APC) labeled, isotype rat IgG antibody. Then the tubes were incubated at4℃30min;250g centrifuged for5min; discarded supernatant, and washed with the FACS buffer2times; resuspended with4%paraformaldehyde solution. Flow cytometry were used to detect CD34+/KDR+positive cells (EPCs). Each105mononuclear cells containing CD34+/KDR+positive cells were counted, the values are expressed in‰.4.2EPCs culturing and identification We extracted20ml peripheral blood from patients, using density gradient centrifugation isolated mononuclear cells. Adherent cells cultured for7d, then Dil-acLDL solution of0.02mg/ml was added and incubated at37℃for2h. Cells were washed with phosphate buffer solution (PBS)2times, fixed by4%paraformaldehyde for15min; washed with PBS2times; added0.01mg/ml FITC-UEA-1solution, incubated at37℃for1h. Then the cells were added10μg/ml4’6-diamidino-2-phenylindole dihydrochloride (DAPI) solution,37℃incubated5min. We observed the cells by inverted fluorescence microscopy, the one that double-positive staining by DiI-acLDL (red) and FITC-UEA-1(green)(double positive cells) were EPCs.7days after EPCs culturing, we randomly selected three vision under a microscope for counting (40times), and calculated the averages.4.3Detecting EPCs migration ability Dulbecco’s modified eagle medium (DMEM) which is containing50ng/mL Vascular endo-thelial growth factor (VEGF) was added to the lower chamber of the modified Boyden chamber. Then adherent cells were digested with2.5g/L trypsin, suspended in DMEM medium.1×105cells suspended per500μl DMEM culture medium were injected into the upper chamber,cultured for24h, then the non-migrating cells were scraped from the membrane. Cells were fixed with methanol, dyed with Giemsa solution. We randomly selected three vision under a microscope for counting the migrating cells (400times), and calculated the averages.4.4Detecting EPCs proliferation ability EPCs were cultured for7days, then were disgested by2.5g/L trypsin, suspended in DMEM medium. EPCs were inoculated into96-well culture plates which coated with human fibronectin in identical amount each well. Each well was added by10μl Methylthiazolyldipheny-tetrazolium bromide (MTT)(5g/L), cultured for4h. Then the supernatant was discarded. The wells were added dimethyl sulfoxide (DMSO)(150μl/well), thoroughly shaken for10min. The absorbance value was measured under a wavelength of490nm in the microplate reader.Results1. Compared the baseline data between intervention group and the drug group: Baseline data showed no significant difference (P>0.05).2. Comparative analysis about Gensini score of the unstable angina patients and the number of EPCs in peripheral blood:the Gensini score and the number of EPCs in peripheral blood was positively correlated (r=0.713, n=56, P<0.01).3. Changes of the number of EPCs in peripheral blood and double-positive cells, cell migration and proliferation capacity post-coronary angiography in the intervention group:3.11week postoperative compared with preoperative The number of EPCs in peripheral blood is higher in1week postoperative than the preoperative (0.57±0.04%o vs0.30±0.04‰); the number of double-positive cells is higher than the preoperative (58.98±10.19vs39.00±4.23); Cell migration capacity is higher than the preoperative (30.82±5.03vs23.71±3.82); proliferative capacity is higher than the preoperative (0.67±0.04vs0.28±0.04). The difference was statistically significant (P <0.01).3.26months postoperative compared with preoperative The number of EPCs in peripheral blood is higher in6months postoperative than the preoperative (0.53±0.04%o vs0.30±0.04%o); the number of double-positive cells is higher than the preoperative (51.05±5.14vs39.00±4.23); Cell migration capacity is higher than the preoperative (29.07±4.41vs23.71±3.82); proliferative capacity is higher than the preoperative (0.52±0.04vs0.28±0.04). The difference was statistically significant (P <0.01).4. Changes of the number of EPCs in peripheral blood and double-positive cells, cell migration and proliferation capacity post-coronary angiography in the drug group:4.11week postoperative compared with preoperative The number of EPCs in peripheral blood is higher in1week postoperative than the preoperative (0.44±0.04%o vs0.30±0.04%o); the number of double-positive cells is higher than the preoperative (58.98±10.19vs39.00±4.23); Cell migration capacity is higher than the preoperative (30.82±5.03vs23.71±3.82); proliferative capacity is higher than the preoperative (0.54±0.04vs0.29±0.03). The difference was statistically significant (P<0.01).4.26months postoperative compared with preoperative The number of EPCs in peripheral blood is higher in6months postoperative than the preoperative (0.45±0.04%o vs0.30±0.04‰); the number of double-positive cells is higher than the preoperative (51.05±5.14vs39.00±4.23); Cell migration capacity is higher than the preoperative (29.07±4.41vs23.71±3.82); proliferative capacity is higher than the preoperative (0.46±0.04vs0.29±0.03). The difference was statistically significant (P<0.01).5. Comparison of the intervention group and drug group post-coronary angiography in the number of EPCs in peripheral blood and double positive cells, cell migration and proliferation capacity5.11week postoperative the number of peripheral blood EPCs is higher in the intervention group than the drug group (56.9±4.2vs43.7±4.0); the number of double-positive cells is higher than the drug group (67.7±4.1%vs48.9±3.6%); the migration capacity is higher than the drug group (34.0±3.4%vs27.1±3.9%); the proliferative capacity is higher than the drug group (0.67±0.04%vs0.54±0.04%). The difference was statistically significant (P<0.01).5.26months postoperative the number of peripheral blood EPCs is higher in the intervention group than the drug group (56.9±4.2vs43.7±4.0); the number of double-positive cells is higher than the drug group (67.7±4.1%vs48.9±3.6%); the migration capacity is higher than the drug group (34.0±3.4%vs27.1±3.9%); the proliferative capacity is higher than the drug group (0.67±0.04%vs0.54±0.04%). The difference was statistically significant (P<0.01).Conclusion1. The severity of coronary lesions and the number of EPCs in the peripheral blood of patients with unstable angina is positively correlated.2. The number of peripheral blood EPCs and double positive cells, cell migration and proliferation ablilty in unstale angina patients are higher1week and6months after drug treatment.3. The number of peripheral blood EPCs and double positive cells, cell migration and proliferation ablilty in unstale angina patients are higher1week and6months after intervention treatment. And compared with pure medication treatment, the increasing magnitude in intervention treatment is more significant. |
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