喉癌Hep-2细胞来源的exosomes的发现和鉴定

目的:观察喉癌Hep-2细胞可否分泌exosomes，并从形态学角度鉴定。方法:大量培养喉癌Hep-2细胞，热休克处理，收集培养上清。先通过3/0.8μm深层过滤小型滤芯对上清进行预处理，去除直径较大的颗粒和杂质。采用Exosome Isolation Kit（商品化试剂盒）收集培养上清液4ml，依次加入Exosome Isolation Kit内试剂，通过exosomes提取专用过滤柱，收集浓缩液。用高倍透射电子显微镜对exosomes做鉴定。结果:成功从喉癌Hep-2细胞培养上清中分离提取出exosomes，电镜观察见exosomes呈圆形或椭圆形双层膜的囊泡状结构，直径约20~100nm，密度较高，分散均匀。结论:首次发现喉癌细胞自身能分泌exosomes，为喉癌免疫治疗做出新的探求。

Discovery and isolation of exosomes derived from laryngocarcinoma Hep - 2 cells

Objective:To observe whether laryngocarcinoma Hep-2 cells can secrete exosomes，and to identify exosomes morphologically.Methods:A large number of laryngocarcinoma Hep-2 cells were cultivated，the yield of exosomes increased by hot shock，cell culture supernatant was gathered.Firstly，the culture supernatant was pretreatment by clarification through a 3/0.8μm small filter element for deep filter to remove particles and impurities with larger diameter.Secondly，exosome isolation kit was used to isolate and extract exosomes.Cells culture supernatant 4ml was gathered，the solutions of the kit were added into the supernatant in proper sequence，then filtered by the special column，the concentrated fluid was obtained.The exosomes were observed under transmission electron microscopy.Results:Exosomes could be isolated and extracted from culture supernatant of laryngocarcinoma Hep-2 cells，and it present circular or elliptical vesicle with bilayer membrane，high density，well-distribution，and with range from 20 to 100nm of diameter.Conclusion:Exosomes can be secreted from laryngocarcinoma Hep-2 cells was first discovered，which provide a new research to laryngocarcinoma immunotherapy.