不同转移习性人鼻咽癌细胞株中外泌体的提取及鉴定

Extraction and identification of exosomes from human nasopharyngeal carcinoma cell lines with different metastatic habits

目的探索不同转移习性人鼻咽癌细胞株SUNE-1-5-8F、SUNE-1-6-10B、SUNE-1和正常鼻咽上皮细胞NP69细胞外泌体的表达情况,为进一步研究鼻咽癌的诊断和治疗新方法提供一定的理论基础。方法在2018年5月—2019年6月期间,用Exosome提取试剂盒从SUNE-1-5-8F、SUNE-1-6-10B、SUNE-1和NP69培养上清液分离出外泌体,使用透射电镜进行观察验证外泌体形态,BCA试剂盒测定外泌体蛋白的浓度,蛋白质印迹法(Western blotting)法进行外泌体特异性抗原分子CD63及筏蛋白-1的检测。结果3种鼻咽癌细胞系和正常鼻咽上皮细胞上清液中均分离出泡状物,在透射电镜下呈椭圆形或者圆形的泡状物。SUNE-1-5-8F、SUNE-1-6-10B、SUNE-1和NP69细胞系外泌体蛋白浓度分别为0.778、0.635、0.788、0.576μg/μL;且所有细胞系来源的外泌体经检测均表达特异性抗原分子CD63及筏蛋白-1。结论3种鼻咽癌细胞株SUNE-1-5-8F、SUNE-1-6-10B、SUNE-1和正常鼻咽上皮细胞NP69细胞均产生外泌体。

Objective To explore the expression of exosomes in human nasopharyngeal carcinoma(NPC)cell lines SUNE-1-5-8F,SUNE-1-6-10B,SUNE-1 and normal nasopharyngeal epithelial cells NP69,and to provide a theoretical basis for further study of new methods of diagnosis and treatment of NPC.Methods Exosomes were isolated from SUNE-1-5-8F,SUNE-1-6-10B,SUNE-1 and NP69 culture supernatants using exosome extraction kits,and verified by transmis-sion electron microscopy.The concentration of exosome protein was determined by BCA kit.Eexosome specific antigen molecule CD63 and Flotillin-1 were detected by western bolt.Results Vesicles were isolated from supernatant of three kinds of nasopharyngeal carcinoma cell lines and normal nasopharyngeal epithelial cells,showed a circular vesicle under a transmission electron microscope.The exosomal protein concentrations of SUNE-1-5-8F,SUNE-1-6-10B,SUNE-1 and NP69 were 0.778,0.635,0.788,and 0.576μg/μL,respectively.CD63 and Flotillin-1 were expressed in all cell line-age-derived exosomes.Conclusion Three kinds of nasopharyngeal carcinoma cell lines SUNE-1-5-8F,SUNE-1-6-10B,SUNE-1 and normal nasopharyngeal epithelial cell line NP69 all produce exosomes.