重度烧伤患者血小板外泌体对凝血功能调节作用的初步研究

Effects of platelet exosomes on coagulation function in severe burn patients:a preliminary study

目的分析重度烧伤患者血小板外泌体对凝血功能调节作用。方法选取内蒙古医科大学第三附属医院烧伤外科6例重度烧伤患者及6例健康志愿者,全部为男性,实验组年龄32~54岁,平均(46±9)岁、对照组年龄28~59岁,平均(48±12)岁,组间年龄差异无统计学意义(P<0.05)。抽取静脉血,高速离心法获取烧伤患者血小板外泌体,通过透射电镜、纳米颗粒分析和Western blot法鉴定。实验组采集烧伤患者血液,对照组采集志愿者,外泌体组为实验组血小板外泌体随机加入对照组血液。通过血栓弹力图(TEG)及常规凝血测试(CCA)比较3组凝血功能。结果外泌体透射电镜下呈典型双层膜类圆形颗粒结构,Western blot法显示标记蛋白TSG101、CD63阳性,颗粒分析显示平均粒径为(120±30)nm,表明细胞外囊泡为外泌体。实验组PT值、APTT值、DD值、R值、K值较对照组升高[两组分别为:16.1(15.0,19.4)s比10.8(10.7,11.1)s、44.7(37.3,53.7)s比28.1(26.6,29.0)s、7.4(6.5,8.9)ng/mL比0.3(0.2,0.3)ng/mL、12.3(10.4,18.3)min比5.8(4.5,7.0)min、3.7(2.9,11.3)min比2.1(1.7,2.3)min,P均<0.05];PLT值、α角、MA值、CI值、LY30值较对照组降低[85.5(70.3,98.5)10^9/L比166.0(157.0,178.0)10^9/L、44.2(22.7,54.1)°比59.6(57.6,61.3)°、46.6(37.9,49.7)mm比56.5(54.0,57.6)mm、-2.1(-3.0,-1.3)比1.3(1.2,1.4)、4.8(1.2,7.9)%比0.0(0.0,0.0)%,P均<0.05]。外泌体组APPT、MA值较对照组升高[25.2(23.9,26.0)s比28.1(26.6,29.0)s、59.4(57.0,61.4)mm比56.5(54.0,57.6)mm,P均<0.05];PT值、R值较对照组降低[10.3(9.9,10.6)s比10.8(10.7,11.1)s、4.4(3.9,5.0)min比5.8(4.5,7.0)min,P均<0.05]。结论TEG检测显示严重烧伤患者休克期后为低凝状态,伴血小板功能降低;严重烧伤患者血小板外泌体在体外实验中起促凝作用。

Objective To analyze the regulating effect of platelet exosomes on coagulation function in patients with severe burns.Methods Six patients with severe burns and six healthy volunteers were selected from the Department of Burn Surgery,the Third Affiliated Hospital of Inner Mongolia Medical University.All of them were male.The age of the experimental group was 32-54 years,with an average of(46±9)years,while the age of the control group was 28-59 years,with an average of(48±12)years.There was no significant difference in age between the two groups(P<0.05).The platelet exosomes of burn patients were obtained by high-speed centrifugation and identified by transmission electron microscopy,nanoparticle analysis and Western blot analysis.The blood of burn patients was collected in the experimental group;that of volunteers collected in the control group;Exosomes group was the experimental group that platelet exosomes were randomly added to the blood of the control group.Thrombelastography(TEG)and conventional coagulation test(CCA)were used to compare the coagulation function of the three groups.Results The exosomes showed a typical double-layer membrane-like circular particle structure under transmission electron microscope.Western blot showed that the marker proteins TSG101 and CD63 were positive.Particle analysis showed that the average particle size was(120±30)nm,indicating that the extracellular vesicles were exosomes.PT value,APTT value,DD value,R value and K value in the experimental group were higher than those in the control group;[16.1(15.0,19.4)s vs 10.8(10.7,11.1)s,44.7(37.3,53.7)s vs 28.1(26.6,29.0)s,7.4(6.5,8.9)ng/mL vs 0.3(0.2,0.3)ng/mL,12.3(10.4,18.3)min vs 5.8(4.5,7.0)min and 3.7(2.9,11.3)min vs 2.1(1.7,2.3)min,all P<0.05].PLT valueαAngle,Ma value,CI value and ly30 value of the two groups were lower than that of the control group[85.5(70.3,98.5)10^9/L vs 166.0(157.0,178.0)10^9/L,44.2(22.7,54.1)°,59.6(57.6,61.3)°,46.6(37.9,49.7)mm vs 56.5(54.0,57.6)mm,-2.1(-3.0,-1.3)vs 1.3(1.2,1.4),4.8(1.2,7.9)%vs 0.0(0.0,0.0)%,all P<0.05].The values of APPT and MA in exosomes group were higher than those in control group;[25.2(23.9,26.0)s vs 28.1(26.6,29.0)s,59.4(57.0,61.4)mm vs.56.5(54.0,57.6)mm,all P<0.05].PT value and R value were lower than those in the control group[10.3(9.9,10.6)s vs 10.8(10.7,11.1)s,4.4(3.9,5.0)min vs 5.8(4.5,7.0)min,all P<0.05].Conclusions TEG detection shows that severe burn patients are in a low coagulation state after the shock phase,accompanied by decreased platelet function;platelet exosomes from severe burn patients play a procoagulant role in vitro experiments.