The Effect And Mechanism Of EPCs Stimulater G-CSF On Cardiac Function In Patients With Heart Failure After Myocardial Infarction

BackgroundEndothelial progenitor cells (endothelial progenitor cells, EPCs) is a primitive cells derived from bone marrow, and human embryonic cells forming blood vessels (angioblast) and human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVEC) is similar to some The conditions can be induced to differentiate into mature endothelial cells. It is not only involved in embryonic angiogenesis, but also after birth, the expression and angiogenesis, vascular endothelial repair and function remains in the still important role. In recent years, the incidence of vascular diseases related to increased year by year. The role of various risk factors, the vascular integrity of cortical damage, deposition of various substances in the blood vessel wall, inflammatory cell infiltration, vascular wall hardening and embrittlement, vascular stenosis is the most basic pathological changes. Current drug treatment and intervention can not be fully restored vascular anatomy, cardiovascular disease or will relapse as time goes on. In vivo tests confirmed, EPCs can differentiate into mature proliferating EPCs, the formation of lumen-like structure, EPCs gradually been applied to treatment of vascular disease treatment, is considered to be vascular disease of the most promising therapeutic measures.In 1977, Asahara and other for the first time reported from isolated human peripheral blood EPCs, thus beginning the true sense of the EPCs of age. Subsequently, Murohara, etc. Also found in cord blood CD34+cells can be induced to differentiate into mature EPCs, that there are umbilical cord blood EPCs. With the continuous development of experimental technology, people have been in the fetal liver, bone marrow, adipose tissue arterial adventitia and isolated EPCs. Experiments confirmed that, EPCs not only when the fetus has an important role in angiogenesis, VEGF expression and angiogenesis after birth, and vascular endothelial maintenance equally important.Myocardial infarction (myocardial infarction, MI) with acute coronary artery occlusion result in some process of myocardial ischemic necrosis. MI surgery, interventional therapy and other traditional therapies are mainly for the reconstruction of the blood and can not fully repair the damage and regeneration of myocardial cells. In recent years, EPCs became the treatment of ischemic cardiovascular disease research focus. Human embryonic EPCs not only involved in angiogenesis, are also involved in postnatal neovascularization (angiogenesis) and endothelial wound repair process. With the understanding of the biological characteristics of EPCs and in the treatment of acute myocardial infarction in the deepening of the application, "mobilization of the mobilization of EPCs from the bone marrow to peripheral blood, participate in the repair of myocardial necrosis and angiogenesis, the prevention of myocardial apoptosis, ventricular remodeling in patients with acute myocardial infarction provides a new way of thinking. The specific mechanism is that when myocardial infarction occurs, a small number of EPCs by the role of inflammatory chemokines was "recruited" to the site of myocardial ischemia, heart and blood vessels involved in regeneration, but this effect is very small, not enough to restore the myocardial injury, it can not prevent the occurrence of left ventricular remodeling and heart failure. In recent years, stem cell mobilization agent in the treatment of myocardial infarction received widespread attention. Applications will be mobilization of EPCs mobilized from bone marrow to peripheral blood, so that the treatment of peripheral EPCs to the quantity needed, the use of its automatic homing (Homing) to myocardial infarction and differentiation characteristics of micro-environment, participation of myocardial necrosis repair and angiogenesis. EPCs have been completed in a number of patients with acute myocardial infarction in the diversity of the animal studies found that a certain level of heart function improved.Cardiac contraction and relaxation are energy-consuming process, myocardial energetics (myocardial energetic, ME), Department of myocardial energy metabolism, aerobic and oxygen balance of the relationship between cardiac work and a discipline. Of myocardial energy metabolism and effective regulation, improved myocardial energy consumption level is the treatment of ischemic heart disease and effective way to inhibit the progression of heart failure. Of myocardial energy metabolism and effective regulation, increased OFR scavenging ability, improved myocardial energy consumption (myocardial energy expenditure, MEE) level, to correct hemodynamic disorders is the treatment of ischemic heart disease and effective way to inhibit the progression of heart failure.EPCs to myocardial infarction combined with cardiac insufficiency treatment opened a new way, but many remain in the animal testing stage, clinical research is still more, the study selected patients with heart failure after myocardial infarction, based on the application of standard treatment G-CSF mobilization of EPCs, observe its effect on inflammatory factors, antioxidant enzymes, changes in myocardial energy consumption, and to explore EPCs possible ways to improve heart function in order to provide a reliable basis for clinical applications.Objects and methods1. Case selection:inclusion criteria①age 75 years of age;②clear past history of myocardial infarction at least 1 month or more;③the gradual emergence of clinical manifestations of heart failure, cardiac function gradeⅡ-ⅣNYHA class;④color Doppler echocardiography Figure confirmed left ventricular dilatation, wall motion abnormalities, resting left ventricular ejection fraction (LVEF)<45%. Exclusion criteria:cardiogenic shock; serious arrhythmias; history of cardiac syncope; liver and renal dysfunction; and hematological malignancy patients with a history.2. All cases cames from PLA 303 Hospital from October 2008 to October 2010.Patients with myocardial infarction were admitted which followed up to meet the inclusion criteria from the nearly 4 months since the onset of internal heart failure patients admitted into the study.24 males and 14 females in all 38 cases, age from 46 to 75 years, mean (60.89±7.53) years old.25 patients with hypertension,20 patients had diabetes mellitus, hyperlipidemia in 21 cases,16 patients who smoke; infarction for 3 months to 2 years, the average (11±8.46) months.9 cases of anteroseptal infarction, anterior infarction in 10 cases,10 cases of extensive anterior wall infarction, inferior wall infarction in 6 cases,3 cases of posterior infarction; preoperative NYHA functional classⅡ/Ⅲ/Ⅳgrade was 10/24/4 cases. On the basis of conventional therapy were randomly divided into treatment and control groups:19 patients in each group. Basic situation of the two groups were balanced in age, gender, complications, infarct site and treatment time, drug use is similar to comparable. Replacement of the 19 healthy volunteers as a health group. 3. Treatment group mobilization on the basis of routine treatment given to mobilizing agent G-CSF (trade name:Kyrgyzstan tablets Fen, Hangzhou Jiuyuan genetic engineering Co, specifications 150μg/support) 450μg/dsc, continuous infusion 5d. The control group received saline injection of the same. All patients in the treatment and follow-up period according to clinical need to give the appropriate drug therapy, including sodium nitroprusside, diuretics, nitrates, antiplatelet drugs, ACEI or angiotensinⅡreceptor blocker (ARB),βreceptor blocking hysteresis agents, statins drugs and blood pressure, reduce blood sugar and other symptomatic treatment.4. EPCs Isolation and identification of:before treatment, respectively, on the mobilization, mobilization of the first 7 days after treatment, patients taking 28 days, 10ml, isolation, culture and determination of the number of EPCs compared before and after treatment the difference between the groups. By density gradient centrifugation from peripheral blood of subjects isolated mononuclear cells were cultured with EBM2 to 7d, analysis of adherent cells. Added to the adherent cells DiLDL, after 1h incubation with 2% paraformaldehyde, and then adding FITC-UEA-I and incubated for 1h, in the inverted fluorescence microscope, the intake of DiLDL combined FITC-UEA-I or double stained positive for differentiating EPCs. The EPCs were cultured in 6 well plate, incubated for 2d not collected adherent cells (including EPCs), adjust the density of 1×106 cells/well into 24-well plates, cultured for 5d, counting each sample in each well number of clones.5. Determination of inflammatory factors:all patients with myocardial infarction patients before treatment and after treatment in 7 days,28 days early in the morning quiet supine elbow vein under fasting blood 10ml, by enzyme-linked immunosorbent assay of plasma high-sensitivity C-reactive protein levels (C-reactive protein, CRP) levels were detected by radioimmunoassay in blood interleukin -6 (interleukin-6, IL-6) and tumor necrosis factor-α(necrotic factor-α, TNF-α) level. High-sensitivity C-reactive protein assay kit by the United States and Biological Products Limited Crystal, Interleukin -6 and tumor necrosis factor-αtest kit from the Beijing Institute of Technology in East Asia to provide immunity. Operate according to kit instructions.6. Determination of antioxidant enzymes:All patients with myocardial infarction patients before treatment and after treatment in 7 days,28 days fasting state early in the morning quiet supine 10ml elbow vein blood anticoagulant,3000r/min, sacrificing the plasma after centrifugation 5min,-80℃kept under test. RBC immediately washed with 0.9% sodium chloride solution 3 times, the SOD extract, to retain the supernatant containing SOD,-80℃preservation. SOD kit by Nanjing Jiancheng Bioengineering Institute, according to the kit procedure. Produced by Japan 200-20 UV spectrophotometer MDA level, PCS from the Nanjing Institute of Biotechnology.7. Plasma BNP concentrations were measured:before treatment, respectively, after treatment 2 weeks and 4 months after treatment, fasting venous blood sample, with 100g/L EDTA anticoagulant, and add aprotinin, plasma was separated after centrifugation using double-antibody sandwich ELISA method for the determination of plasma BNP concentration.8. At the same time determination of blood lipids, blood, blood glucose, liver and kidney function, serum creatine kinase and troponin and other biochemical markers. CK, CK-MB kits were provided by the German MAN Bowring, with kinetic assay, the instrument for the Italian production of CB-171 semi-automatic biochemical analyzer.9. Cardiac function and myocardial energy consumption test:The GE Logig 7 color Doppler ultrasonography, respectively, on admission,2 weeks after treatment and 4 months, routine echocardiography, measuring left ventricular long axis view left ventricular short axis view, apical four chamber view to measure left ventricular structure indicators such as:ejection fraction (EF), fractional shortening (FS), left ventricular end systolic diameter (LVIDs), left ventricular end-diastolic volume (EDV), end systolic left ventricular posterior wall thickness (PWTs), stroke volume (SV), ejection time (ET), the indicators from 3 consecutive cardiac cycles on average. The end of the ultrasound examination, recorded the patient's height, weight, systolic blood pressure measured by cuff sphygmomanometer. Reference by the relevant formula:left ventricular end-systolic circumferential wall stress (circumferential end-systolic wall stress, cESS) on behalf of left ventricular end systolic measure of tension: , MEE (non-invasive method to evaluate the myocardial bio-energy consumption):MEE (cal/systole)= cESS (kdyn/cm2)×ET(s)×SV(ml)×4.2×10-4. Clinical evaluation criteria standards by NYHA functional class, heart failure or heart function improved to correct for more than two markedly; cardiac function level is effective; no improvement or deterioration of cardiac function were invalid.10. Statistical Methods Statistical analysis:All data were processed using statistical software SPSS13.0. Measurement data with mean±standard deviation. Between the two are used to compare the number of two independent samples t test was used to compare the mean number of repeated measures analysis of variance, testing the level of bilateral, associated with Pearson correlation analysis method to P <0.05 was considered statistically significant.Results1. Safety effects of two groups of patients have to mobilize more significant treatment group; 38 cases of myocardial infarction patients were safe, no serious complications after treatment follow-up. The study found that patients with myocardial infarction complicated by heart failure in the process of G-CSF, the most common adverse events were fever, G-CSF group did not find adverse cardiovascular events such as arrhythmia, heart failure progression and recurrence myocardial infarction.2. G-CSF injection changes in the number of peripheral blood EPCs:myocardial infarction patients with an average of EPCs than healthy volunteers of water (P <0.01); EPCs after treatment, blood levels were higher than before treatment (P <0.05), in addition to the control group 28 days after treatment, no significant difference (P>0.05); treatment group, G-CSF EPCs in peripheral blood cells increased in the drug reached a peak on day 7, compared with the control group had significantly higher (P<0.01).3.G-CSF injection of BNP levels before and after:After treatment, plasma BNP levels were decreased compared with before treatment and returned to normal, the difference was statistically significant (P<0.01); the treatment group and 28 after 7 days days of plasma BNP levels lower than the control group, were significantly different (P<0.05), after treatment both groups showed no significant difference at different times (P>0.05).4. Inflammatory factor test results:Myocardial infarction complicated by heart failure patients CRP, TNF-α, IL-6 increased significantly compared with healthy volunteers (P<0.05). Compared with before treatment, after treatment, serum CRP, TNF-α, IL-6 decreased, with statistical significance (P<0.05), the 7th day treatment group than in the control group CRP decreased significantly (P<0.01). Determination of antioxidant enzyme activity Results:The patients were treated by the improved myocardial energy metabolism, heart tissue SOD activity increased, LPO, MDA formation decreased (P<0.05), changes in the treatment group than the control group, there was differences (P<0.05).5. Echocardiography results:two groups of cardiac function after treatment than before treatment were improved, LVIDs, PWTs, cESS, MEE than those before treatment, ET, SV, LVEF, FS increased compared with before treatment (P<0.05); changes in the treatment group than the control group significantly (P<0.05); especially in the treatment group cESS, MEE 2 weeks had significantly decreased (P <0.01); and the treatment group LVEF, FS Section 4 months had significantly increased (P<0.01); EDV and compared with the control group before treatment and no significant difference (P> 0.05).6. Comparison of the efficacy of cardiac function in 19 patients in the control group,1 case of markedly effective in 14 cases,4 cases, total effective rate 78.9%; treatment group 2 patients, effective in 15 cases,2 cases, the total effective rate 89.4%. The total effective rate between the two groups had significant difference (P <0.05).Conclusion1.G-CSF mobilization of EPCs combined treatment of MI in patients with heart failure can improve cardiac function, and its mechanism may be mobilized to increase the number and function of EPCs were effective in improving left ventricular ejection fraction, reduce cardiac stress, the heart blood hemodynamics and myocardial energy consumption to adjust and improve the effect.2.G-CSF mobilization of EPCs in patients with heart failure after treatment of myocardial infarction, but also inhibition of CRP, TNF-α, IL-6 expression and generation of inflammatory cytokines regulate the anti-inflammatory cytokines and the expression of pro-inflammatory cytokine balance, which inhibition of cardiac tissue and the local inflammatory reaction and improve heart failure after myocardial infarction "cachexia" state. 3. EPCs have the protection of ischemic myocardium, enhanced SOD activity, inhibition of LPO, MDA generation and reduce peroxidation, increase the antioxidant capacity of patients with heart failure and the ability to eliminate oxygen free radicals, reduce myocardial damage to improve heart function.