白血病细胞来源外泌体的提取和鉴定

Extraction and identification of exosomesderivedfrom leukemia cells

目的:从白血病细胞系HL-60、THP-1中分离外泌体,并对HL-60所得外泌体进行鉴定。方法:使用Total Exosome Isolation(invitrogen)试剂盒法从白血病细胞系HL-60、THP-1培养上清液中分离出外泌体,并使用透射电镜观察其形态特征,ZETASIZER Nano series-Nano-ZS仪分析外泌体粒径大小,流式细胞术进行外泌体特异性抗原分子CD63及CD81鉴定。结果:2种白血病细胞系培养上清液中均分离出大量泡状物,透射电镜下观察外形呈圆形或椭圆形杯状囊泡,胞膜完整,内含低电子密度物质;粒径分析其直径为30~150nm,且此种囊泡状物表达外泌体特异性蛋白CD63、CD81。结论:从2种白血病细胞上清中分离的囊泡状物质为外泌体,Total Exosome Isolation(invitrogen)法不需要超速离心,能够简便快速地从白血病细胞系培养上清液中提取到外泌体,为外泌体在白血病中的诊断和治疗提供了新的研究方法和理论依据。

Objective To isolate and identify exosomes from leukemia cell lines HL-60 and THP-1. Methods Isolate exosomes from leukemia cell line HL-60 and THP-1 cultural supernatant using the Total Exosome Isolation(invitrogen) kit, and identify exosomes by observing the morphological characteristics with transmission electron microscope, analysis the diameter size of exosomes using ZETASIZER Nano series-Nano-ZS instrument, detect the specific antigen molecules of exosomes CD63 and CD81 by flow cytometry. Results A large number of vesicles were isolated from 2 kinds of leukemia cell line cultural supernatant, which appears round or oval cup shape, with completed membrane and contains low electron density materials under transmission electron microscopy vision, ranges from 30-150 nm in diameter size, and expresses the specific antigen molecules of exosomes CD63 and CD81. Conclusion The vesicles isolated from the two kinds of leukemia cells cultural supernatant are exosomes, and Total Exosome Isolation method(invitrogen) can easily and quickly extract exosomes from leukemia cell lines without overspeed centrifugal, this provides a new method and theoretical basis for the diagnosis and treatment of exosomes in leukemia.