喉癌Hep-2细胞来源的exosomes提取方法的比较  

Comparison of methods for isolating exosomes derived from laryngocarcinoma Hep-2 cells

作　　者：梁俊毅[1] 吉晓滨[1] 刘启才[2] 谢景华[1] 

机构地区：[1]广州市第一人民医院耳鼻咽喉科,广州510180 [2]广州医科大学实验医学研究中心

出　　处：《临床耳鼻咽喉头颈外科杂志》2015年第17期1522-1526,共5页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：广东省科技厅产业技术研究与开发资金计划项目(No:2012B031800340);广州市科技和信息化局社会发展应用基础研究专项(No:2013J4100024)

摘　　要：目的:对分离提取喉癌Hep-2细胞来源的exosomes 2种方法进行对比,展现不同方法的优缺点,为选择分离提取包括喉癌Hep-2细胞在内的肿瘤细胞来源exosomes的方法提供参考。方法:大量培养喉癌Hep-2细胞,42℃热休克处理。收集90ml培养上清液,先通过3/0.8μm深层过滤小型滤芯对上清液进行预处理,去除直径较大的颗粒和杂质,再利用蔗糖密度梯度离心联合超滤离心法,将上清液浓缩提纯;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2种方法所得的exosomes浓缩液分别鉴定,作出评价。结果:2种方法均能成功地从喉癌Hep-2细胞培养上清液中分离提取出exosomes。单个高倍视野下,蔗糖密度梯度离心联合超滤离心法提取exosomes分散性较好,但密度较低,背景见杂质较多;Exosome Isolation Kit所提取exosomes排列紧密,密度较高,背景杂质较少。结论:2种方法各具特点,均是较理想的exosomes分离提取方法。利用Exosome Isolation Kit分离提取exosomes具有样本量少、提取时间短、步骤简单、产物量大等优点,为喉癌Hep-2来源exosomes的分离提取提供了较好的选择。Objective：To provide a reference about choosing the methods of isolating exosomes derived from tumor ceils including laryngocarcinima Hep-2 cells by comparing advantages and defects of two methods of isola- tion and extraction exosomes. Method：Previously, laryngocarcinoma Hep-2 ceils were cultivated massively, then the cells were processed with hot shock in 42℃ for 1 h. Sucrose density gradient centrifugation ultrafiltration （method 1） ： cells culture supernatant 90 ml was gathered,the supernatant was clarified through a 3/0.8μm small filter to remove impurities and fragments which in larger diameter. Then the filtering fluid was concentrated and purified through sucrose density gradient centrifugation and ultrafiltration, the concentrated fluid was obtained. Exosome Isolation Kit （method 2） ：cells culture supernatant 4 ml 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. Both products are observed by high resolution transmission electron microscopy. Result：Both methods could isolate and extract exosomes feasibly. In single high power view of transmission electron microscopy, exosomes of method 1 disperse better, but lower density, and more impurity in background, exosomes of method 2 arrange closer, higher density, and less impurity. Conclusion： Exosome Isolation Kit require less supernatant, cost less time, process proce- dure briefly,harvest higher yield. It may become a new option of isolating exosomes derived from Laryngocarcino- ma Hep-2 cells.

关 键 词：EXOSOMES 蔗糖密度梯度离心 超滤离心 EXOSOME ISOLATION KIT 

分 类 号：R739.6[医药卫生—肿瘤]