保存期间血小板中外泌体浓度变化及其对凝血功能的影响  

Changes of exosomal concentration during platelet storage and the effects on coagulation function

作　　者：龚丽 米紫玥 孔玉洁[2,3] 徐海霞 田力 刘忠 Gong Li;Mi Ziyue;Kong Yujie;Xu Haixia;Tian Li;Liu Zhong(School of Public Health,Anhui Medical University,Hefei 230032;Institute of Blood Transfusion,Chinese Academy of Medical Sciences and Peking Union Medical College,Chengdu 610052;Key Laboratory of Transfusion Adverse Reactions,Chinese Academy of Medical Sciences,Chengdu 610052)

机构地区：[1]安徽医科大学公共卫生学院,合肥230032 [2]中国医学科学院/北京协和医学院输血研究所,成都610052 [3]中国医学科学院输血不良反应研究重点实验室,成都610052

出　　处：《安徽医科大学学报》2022年第8期1283-1288,共6页Acta Universitatis Medicinalis Anhui

基　　金：中国医学科学院中央级公益性科研院所基本科研业务费专项资金(编号:2018PT32016);四川省国际科技创新合作/港澳台科技创新合作项目(编号:2020YFH0024)。

摘　　要：目的探讨不同保存时间手工血小板中外泌体浓度变化及其对凝血功能的影响。方法等体积分装血小板,(22±2)℃保存0、3、5 d(D 0、D 3、D 5)后,超速离心法提取血小板外泌体;采用Western blot、透射电子显微镜、纳米流式分析仪对所提外泌体进行表征鉴定。使用纳米流式分析仪检测其颗粒浓度。取不同保存时间血小板外泌体至人新鲜血浆中,全自动凝血分析仪检测血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)。通过Western blot实验进行凝血因子VII含量测定。取不同保存时间血小板外泌体至人新鲜全血中,血栓弹力图仪检测外泌体对全血凝血指标的影响。结果鉴定结果显示外泌体免疫标志物TSG 101、CD 9特异性表达;所提外泌体呈现双层膜、类杯状结构;其粒径多为60~80 nm之间。D 0、D 3、D 5外泌体浓度分别为(13.86±7.93)×10^(12)、(23.69±12.80)×10^(12)、(45.81±25.87)×10^(12)个/L。与D 0相比,D 3、D 5外泌体浓度显著增加(P<0.01),分别为1.8倍、4.0倍。与对照组相比,D 0与D 5外泌体分别可使PT减小[(0.16±0.15)s,P<0.01]、[(0.36±0.18)s,P<0.001];D 0与D 5外泌体分别可使APTT减小[(1.11±1.21)s,P<0.05]、[(2.10±1.14)s,P<0.001]。凝血因子Ⅶ定量实验结果显示其在D 0、D 5外泌体中含量差异有统计学意义(P<0.05)。血栓弹力图实验结果显示对照组、D 0、D 5外泌体作用于全血后其凝血时间R值分别为(5.46±0.90)min、(5.00±0.67)min、(4.38±1.00)min,D 5组与对照组、D 0组差异有统计学意义(P<0.05),提示外泌体具有一定的促凝作用。结论随着保存时间延长,手工血小板中外泌体浓度显著增加且外泌体的促凝作用显著增强,血小板保存期间产生的外泌体可能通过凝血因子Ⅶ参与促凝过程。Objective To detect exosomes concentration changes in manual platelet with different storage time and further investigate the effects of exosomes on coagulation function.Methods After divided into storage bags with same volume,the platelets were stored for 0,3,5 days(D 0,D 3,D 5)in(22±2)℃.Exosomes were isolated from platelet samples by ultracentrifugation.Western blot、transmission electron microscope and nano-flow cytometry were adopted to characterize exosomes.Meanwhile,exosomes particles concentration was detected by nano-flow cytometry.Exosomes with different storage time were added to plasma,then the effects on the plasma prothrombin time(PT)and activated partial thromboplastin time(APTT)were detected by the automatic coagulation analyzer.Expression of coagulation factor VII on exosomes from platelet stored for different time were tested by Western blot.Exosomes samples of D 0,D 5 were incubated with fresh whole blood,respectively.Then the coagulation indexes of each samples were detected by Thromboelastography test(TEG).Results Test showed the presence of TSG 101,CD 9,which were exosome specific markers.And the exosomes showed a double-layer membrane and cup-like structure.Moreover,the particle size distribution of exosomes was mainly between 60~80 nm.The concentration of exosomes stored for D 0,D 3,and D 5 were(13.86±7.93)×10^(12),(23.69±12.80)×10^(12),(45.81±25.87)×10^(12)particles/L,respectively.Compared with D 0,the concentration of exosomes stored for D 3 and D 5 increased significantly(P<0.01),with 1.8 times and 4.0 times,respectively.Compared with the control group,PT in the D 0 and D 5 exosomes group could be decreased by[(0.16±0.15)s,P<0.01]、[(0.36±0.18)s,P<0.001],APTT could be decreased by[(1.11±1.21)s,P<0.05]、[(2.10±1.14)s,P<0.001],respectively.Semi-quantitative analysis of coagulation factor VII was performed by Western blot and it showed statistically differences in coagulation factorⅦconcentration(P<0.05).TEG test showed that the coagulation time R value of control group,ex

关 键 词：血小板 外泌体 凝血 凝血因子Ⅶ 

分 类 号：R331.143[医药卫生—人体生理学]