Study On The Microparticles Of Peripheral Blood In Patients With Coronary Heart Disease And Blood Stasis Syndrome

Purpose:We optimized the method of detecting the microparticles by Flow cytometry and found the differences between the level of RMPs、 LMPs、PMPs and EMPs. In this way we found a potential predictor of coronary heart disease. Then, we further researched the changes between the unstable angina pectoris and stable angina pectoris as well as the differences between blood stasis syndrome and non-blood stasis syndrome. So as to reveal the formation and pathogenesis of coronary heart disease from the perspective of the microparticles and provide new ideas of coronary heart disease in clinical studies.Methods:We selected 120 CHD patients from March 2014 to February 2015 in Guang’anmen Hospital, used 31 healthy volunteers from Southern District of Guang’anmen Hospital as a control group. The coronary heart disease patients were divided into UA and SA group, as well as blood stasis syndrome group and non-blood stasis syndrome group. All subjects’ blood were save by sodium citrate anticoagulation. Using gradient centrifugation method for preparing platelet-free plasma, the PFP were kept in-80 ℃ refrigerator and tested in two weeks by flow cytometry. The four sources of microparticles were divided into 18 different projects according to size and labeled, analyzed the correlation between the value of coronary heart disease, and found the potential indicators of coronary heart disease by ROC analysis. In the methodology part, we used unstable angina plasma to investigate the level of micropar-ticles in different centrifugal force and centrifugation time and concluded an effective way to get the microparticles.Results:1 Under the centrifugal force of 13000g, for SMPs, different centrifugation time:3min> 6min (P＜0.01),6min> 10min (P<0.01),10min> 15min (P＜0.01); for BMPs:3min> 6min (P<0.01),6min> 10min (P＜0.01),10min> 15min (P<0.05) and for platelets,3min> 6min (P<0.01),6min> 10min (P<0.01),10min> 15min (P<0.05). In centrifugation time of 30min, different centrifugal force:16000g<18000g<20000g<22000g. There was significant difference between 18000g and 20000g.2 UA with plasma SMPs were significantly more than UA with BMPs (P＜0.01); the number of MP’s by single-labeled were significantly more than the number of multi-labeled.3 SMP{AV+), SRMP(CD235+), SPMP(CD41a+), SLMP(CD45+), SRMP(AV+/CD 235+), SPMP(AV+/CD41a+), SLMP(AV+/CD45+), SEMP(AV+/CD144+), BMP(AV+), BRMP(CD235+), BPMP(CD41a+), BLMP(CD45+), BEMP(CD144+), BRMP(AV+/CD235 +), BPMP(AV+/CD41a+), BLMP(AV+/CD45+), BEMP(AV+/CD 144+) in CHD plasma was significantly higher than the healthy control group (P<0.05 or P<0.01) and they were positively correlated with coronary heart disease(P<0.05 or P<0.01); BPMP(AV+/CD41a+), SLMP(AV+/CD45+), BMP (AV+), SMP(AV+) in each group had the most significant independent risk factor for coronary heart disease by logistic regression and their area under the roc curve were 0.79,0.732,0.777,0.676. The threshold of CHD were 472counts/nl, 530counts/μ1,462counts/μ1,922counts/μ1.4 Unstable Angina group:SMP(AV+), SRMP(CD235+), SPMP(CD41a+)、 SLMP (CD45+), SRMP (AV+/CD235+), SPMP (AV+/CD41a+), SLMP(AV+/CD45+), BMP(AV+), BRMP(CD235+), BPMP(CD41a+), BLMP(CD45+), BEMP(CD144+)^ BRMP(AV+/CD235 +),BPMP(AV+/CD41a+),BLMP(AV+/CD45+),BEMP(AV+/CD144+) levels were significant-ly higher than SA group (P<0.05 or P<0.01); In addition to BEMP (AV+/CD144+), the others were positively correlated with coronary heart disease (P<0.05 or P<0.01).5 Blood stasis syndrome:SMP(AV+), SRMP(AV+/CD235+), SPMP (AV+/CD41a+), SLMP(AV+/CD45+), BMP(AV+), BRMP(CD235+), BPMP(CD41a+), BLMP(CD45+), BEMP(CD144+), BRMP(AV+/CD235+), BPMP(AV+/CD41a+), BLMP(AV+/CD45+) were significantly higher than non-blood stasis syndrome group(P<0.05 or P<0.01), and they were all positively correlated with coronary heart disease (P<0.05 or P<0.01).Conclusion:1 It relatively had few platelets under the condition of 13000g 10min in the microparticles-rich plasma, and it could effectively prevent producing more microparticles by cryopreservation or platelet activation; Microparticles were basically precipitated under the condition of 20000g 30min which could not only clean the unbound antibody, but also avoid the added activation of the coagulation system by adding the Anniex V and binding buffer and it could be a effective method of extraction and purification of microparticles.2 UA with plasma SMPs significantly more than UA with BMPs (P<0.01); the number of MP’s by single-labeled significantly were more than the number of multi-labeled.3 The number of RMPs, PMPs, LMPs, EMPs were rise in CHD patients’plasma; SMP(AV+)、BMP(AV+)、BPMP(AV+/CD41a+)、SLMP(AV+/CD45+) in each group were the strongest role in the diagnosis of coronary heart disease, Their threshold of CHD were 922counts/μ1,462counts/μ1,472counts/μ1,530counts/μ1; They all had better sensitivity and specificity in the diagnosis of coronary heart disease and BPMP(AV+/CD41a+) was the strongest diagnostic indicator of coronary heart disease.4 The number of RMPs, PMPs, LMPs, EMPs rose in Unstable Angina pectoris patients’ plasma and they were positively correlated with UA. It showed that coronary heart disease was closely related to microparticles.5 The number of RMPs, PMPs, LMPs, EMPs rose in blood stasis syndrome group patients’plasma and they were positively correlated with CHD stagnatioin syndrome. It showed that microparticles were predictors and reference indicator of diagnosis and treatment of chinese traditional medicine.