单采血小板来源外泌体的提取与鉴定

The Extraction and Identification of Apheresis Platelet-derived Exosomes

目的研究单采血小板来源外泌体提取和鉴定的常见影响因素,为后续开展单采血小板来源外泌体研究提供参考。方法利用超速离心法分离提取单采血小板来源外泌体,并通过透射电镜、Western Blotting验证提取产物;利用BCA试剂盒测定外泌体总蛋白浓度。结果 5天组外泌体蛋白浓度(1822.2±11. 4μg/mL)明显高于1天组(775. 7±11. 5μg/mL),P <0.001;未冻存组蛋白浓度(1822.2±11.4μg/mL)明显高于不含DMSO冻存组(1153.6±2.6μg/mL)和含5%DMSO冻存组(1403.4±6.1μg/mL),P<0. 001。结论单采血小板来源外泌体含量随标本保存时间延长逐渐增加;冻存处理会一定程度上降低外泌体提取产量和影响其形态观察。

Objective To study the common influencing factors of the exosomes extraction and identification from apheresis platelet,so to provide reference to the follow-up study of platelet-derived exosomes. Methods We separated and extracted the exosomes from apheresis platelet and verified them by transmission electron microscope and western blotting;. The protein concentration of exosomes was then determined by BCA kit. Results The protein concentration of the exosomes in 5-day group was 1822.2±11.4 μ g/mL ,while that of the 1-day group was 775.7±11.5 μ g/mL, P <0.001;The protein concentration of exosomes in untreated group was 1822.2±11.4 μ g/mL,which was significantly higher than that of cryopreservation group without DMSO (1153.6±2.6 μ g/mL)and cryopreservation group with 5% DMSO (1403.4±6.1 μ g/mL), P < 0.001 . Conclusion The content of exosomes from apheresis platelet will increase gradually with the prolongation of the specimen preservation time;The cryopreservation process will decrease the yield of exosomes and affect the morphological observation to certain extent.