大鼠T细胞源性exosome提取方法比较  

The comparison among extraction methods of exosomes from T cells of rats

作　　者：孙祯[1] 黄赤兵[1] 宋亚军[1] 陈益荣[1] 李传贵[1] 

机构地区：[1]第三军医大学新桥医院泌尿外科,重庆400037

出　　处：《重庆医学》2013年第29期3512-3514,3517,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(30872580)

摘　　要：目的采用3种方法从大鼠T细胞培养上清中提取纯化exosome,以获取高质量的exosome。方法分别采用ExoQuick Precipitation提取法、超滤密度梯度离心法、差速离心法提取T细胞培养上清中的exosome。利用透射电镜进行形态学观察,2,2-联喹啉-4,4-二甲酸二钠(BCA)法进行蛋白定量,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析蛋白表达的差异,Western blotting检测白细胞介素2(IL-2)的表达。结果 3种方法均可提取出exosome;ExoQuick Precipitation提取法、超滤密度梯度离心法所得样本浓度显著高于差速离心法所得样本浓度(P<0.05);SDS-PAGE结果显示3种方法所得样本具有蛋白表达强度的差异;Western blotting显示3种方法所得样本均表达IL-2。结论 ExoQuick Precipitation提取法、超滤密度梯度离心法可获得高纯度、无蛋白丢失的exosome样本。Objective The purification methods of the exosomes derived form T cells were established in order to get high quan-tity exosomes .Methods Exosomes from T cells culture supernatants were purified by ExoQuick Precipitation ,ultrafiltration and sucrose gradient centrifugation ,differential ultracentrifugation ,and confirmed via using transmission electron microscopy .The pro-tein expression of the exosomes were analyzed by SDS-PAGE electrophoresis .Western blotting was used to test the expression of IL-2 .Results The protein concentration of the exosomes purified through ExoQuick Precipitation ,ultrafiltration and sucrose gradi-ent centrifugation were higher than through differential ultracentrifugation （P〈0 .05） .SDS-PAGE displayed the difference among the exosome purified by three methods .Three kinds of exosomes all expressed IL-2 .Conclusion ExoQuick Precipitation ,ultrafiltra-tion and sucrose gradient centrifugation technique can obtain high purity and complete exosome sample .

关 键 词：EXOSOME ExoQuick PRECIPITATION 超滤密度梯度离心 差速离心 

分 类 号：R-332[医药卫生]