The Relationship Between Plasma Exosomes Of Acute Myocardial Infarction Patients And Myocardial Enzymes And The Degree Of Coronary Artery Disease

Objective: In this study, we investigate quantitative and qualitative analysis of plasma derived Exosomes of patients with acute myocardial infarction, discussing about the correlation between Exosomes and plasma enzyme concentration, coronary artery disease. Learn more about the level of peripheral blood derived Exosomes in the development of myocardial infarction and its effect on the prediction of acute myocardial infarction and the value of the application.Methods: 1.Clinical patient selection: Coronary angiography (CAG) was performed in 93 hospitalized patients who were suspected as having CAD.The patients were categorized as Group N (n = 24, control group), Group AMI (n =23, acute myocardial infarction), Group SA (n = 24, stable angina pectoris) and Group UA(n = 22, unstable angina pectoris). 2. Isolation of plasma Exosomes: Obtain 40 ml blood from patients undergoing coronary angiography, using the ficoll method to get plasma, centrifugate 30 min at 2,000×g, 4?C.Carefully transfer supernatant to ultracentrifuge tubes or bottles without pellet contamination. Centrifuge 45 min at 12,000×g, 4?C, carefully transfer supernatant to ultracentrifuge tubes or bottles . Centrifuge 2 hr at110,000×g, Resuspend pellets in 1 ml PBS and pool them in one of the tubes. Fill the tube with PBS to dilute the resuspension in a large volume. Filter the suspension through a 0.22-μm filter, and collect in a fresh ultracentrifuge tube or bottle. Centrifuge 70 min at 110,000×g, 4?C. Pour off the supernatant. Resuspend the pellet in 1 ml PBS, and then fill the tube with PBS. Centrifuge 70min at 110,000×g, 4?C. Pour off the supernatant. Resuspend pellet in 50 to 200μl PBS. Store up to 1 year at ? 80?C.3.BSA Protein analysis:Exosomes 2μl,dilute with PBS for 5 times,follow the manufacturer manual, according to substrate ratio of 1:20 with the sample,37 centidegree water bath for 30 minutes, analysis on the Nanodrop machine for BSA protein .4. Analysis of Exosomes by FACS of labeled Exosomes bound to beads: Incubate 5μg purified Exosomes as measured by BSA protein Add PBS to a final volume of 1 ml, and incubate on a test tube rotator wheel 2 hr at room temperature. kit with 10μl latex beads 15 min at room temperature, in a 1.5 ml microcentrifuge tube. Add 110μl of 1 M glycine (i.e., 100 mM final), mix gently and let stand on the bench at room temperature for 30 min. Microcentrifuge 3 min at 4000 rpm, room temperature. Remove the supernatant and discard. Resuspend the bead pellet and microcentrifuge as in step 5 to wash two more times. Resuspend beads in 0.5 ml PBS/0.5% BSA. Incubate 10μl coated beadswith hycoerythrin (PE)-CD11c,pacific bule–CD86,APC/cy7-MHC-DR,FITC-CD1a,PE-CD54,PE-CD106 ,FITC-CD3 and the corresponding isotype-matched antibodies[1]. 5. Lipids, HbA1c,CK,CK-MB,CK-MM,FPG, blood pressure, age, sex and prior medical histories including hypertension, diabetes mellitus and smoking status were obtained before CAG in all patients.The severity of pathological changes of the coronary anery was assessed by the number of diseased coronary branlches and Gensini's score.6.All data were analysised by software SPSS 18.0. Measurement data were presented as mean±standard deviation ( x±s) . Diffrences between continuous variables were tested for statistical significance by means of t tests, one-way anlalysis -of- variance or Kruskal-Wallis H test (multiple groups), and proportions were tested byχ2 test. We assessed associations among Exosomes and CK,Gensini scores using speannan's correlation coeffcient.Risk factors for coronary heart disease using binary Logistic regression analysis. Gensini scoring was the dependent variable of the regression model. And age, total cholesterol, triglycerides, low density lipoprotein, high density lipoprotein, WBC count, CD11C,CD86,MHC-DR ,CK,CK-MB,CK-MM were the independent variable. P<0.05 was considered statistically significant.Results:①The level of Exosomes protein of AMI and UA was significantly higher than that in controls,AMI Exosomes was significantly highly than SA.From FACS result, the level of CD11C, CD86 and MHC-DR was significantly higher than that in controls.②The protein level of plasma Exosomes and Gensini score in AMI and UA patients was significantly higher than that in SAP patients, indicted Exosomes may related to the stability of coronary lesions.③Plasma Exosomes level was positively correlated with the level of CK,CK-MB,CK-MM,WBC。④Plasma Exosomes level increased with the increasing of Gensini's score, it means that Exosomes also related to the severity coronary artery.Conclusions: This study demonstrates that increased plasma Exosomes level is associated with the occurred and Severity of AMI .Plasma Exosomes levels was positively related to plasma CK level, Gensini points, it was the independent risk factor of Gensini score. In addition plasma levels of CK and coronary angiography, plasma Exosomes level may be to assess the acute myocardial infarction clinical indicators.