非小细胞肺癌A549细胞培养上清液中外泌体的分离提取及鉴定  

Isolation,extraction and identification of exosomes from non-small cell lung cancer A549 cell culture supernatant

作　　者：郑巧鑫 苏欣 魏淑贞 刘诗怡 罗晶 黄颖 苏会岚 李娜 ZHENG Qiaoxin;SU Xin;WEI Shuzhen;LIU Shiyi;LUO Jing;HUANG Ying;SU Huilan;LI Na(School of Public Health,Chengdu Medical College,Chengdu 610500,Sichuan Province,China)

机构地区：[1]成都医学院公共卫生学院,四川成都610500

出　　处：《中国生物制品学杂志》2023年第11期1368-1372,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81401757);成都医学院自然科学基金(CYZYB21-02);成都七院合作项目(2021LHJYPJ-05)。

摘　　要：目的分离提取非小细胞肺癌A549细胞培养上清液中的外泌体,并进行鉴定。方法分别采用超速离心法(ultracentrifugation,Ult)及改良法[即Ult法结合试剂盒Exo QuickTM(Ult-Exo QuickTM,U-EQ)]分离提取非小细胞肺癌A549细胞培养上清液中的外泌体。BCA法测定外泌体浓度,纳米粒子追踪分析(nanoparticle tracking analysis,NTA)检测外泌体粒径分布,荧光染色及透射电镜(transmission electron microscope,TEM)法观察外泌体的形态,Western blot法检测外泌体表面CD63和HSP70蛋白的表达情况。结果U-EQ及Ult法分离提取外泌体的浓度分别为1.355和0.909 mg/mL;颗粒总数分别为4.65×10^(9)和4.06×10^(9)粒/mL,峰值尺寸分布范围分别为81.5~165.5和59.5~176.5 nm,平均尺寸分别为(152.3±8.8)和(94.3±9.8)nm;直径均为30~150 nm,U-EQ法分离提取外泌体的形态更均一;均可见CD63和HSP70蛋白表达,U-EQ法分离提取的外泌体蛋白表达量更高。结论U-EQ及Ult法均可从非小细胞肺癌A549细胞培养上清中分离提取外泌体,U-EQ法提取外泌体的浓度更高,粒径更均一。Objective To isolate,extract and identify exosomes from the culture supernatant of non-small cell lung cancer A549 cells.Methods Exosomes from supernatant of non-small cell lung cancer A549 cells were isolated by ultracentrifugation(Ult)and modified method[Ult combined with Exo Quick~(TM)(Ult-Exo Quick~(TM),U-EQ)],which were determined for the concentration by BCA,measured for the particle size distribution by nanoparticle tracking analysis(NTA),observed for the morphology by fluorescence staining and transmission electron microscope(TEM),and detected for the expression of CD63 and HSP70 proteins on the surface of exosomes by Western blot.Results The concentrations of exosomes isolated by U-EQ and Ult were 1.355 and 0.909 mg/mL,respectively;The total number of particles was 4.65×10~9 and 4.06×10~9 particles/mL,the peak sizes ranged from 81.5 to 165.5 and 59.5 to 176.5 nm,and the average size was(152.3±8.8)and(94.3±9.8)nm,respectively;The exosomes were all 30~150 nm in diameter,while the morphology of exosomes extracted by U-EQ method was more uniform;CD63 and HSP70 proteins were expressed in all of them,while the exosomes extracted by U-EQ method showed higher expression.Conclusion Both U-EQ and Ult methods can isolate exosomes from the supernatant of non-small cell lung cancer A549 cells,while the concentration of exosomes extracted by U-EQ method is higher.

关 键 词：非小细胞肺癌A549细胞 外泌体 分离提取 

分 类 号：R111[医药卫生—公共卫生与预防医学]