肿瘤细胞来源胞外体的分离鉴定与功能检测  

Separation and Identification of the Exosomes Derived from A Human Tumor Cell Line

作　　者：崔焱[1] 于津浦[2] 李慧[2] 杨莉莉[2] 魏枫[2] 安秀梅[2] 任秀宝[2] 

机构地区：[1]天津医科大学附属肿瘤医院高级病房,300060 [2]天津医科大学附属肿瘤医院免疫室,300060

出　　处：《天津医药》2009年第12期1014-1016,I0007,共4页Tianjin Medical Journal

摘　　要：目的:分离提纯和电镜鉴定乳腺癌细胞系MCF-7细胞分泌的胞外体,并观察其对淋巴细胞增殖的影响。方法:将细胞培养上清进行分步离心、浓缩、密度梯度离心获得纯化后的胞外体,并对胞外体进行透射电镜鉴定。将分离纯化的胞外体加入含有IL-2的淋巴细胞培养体系中,用MTT试验计算淋巴细胞的增殖率,观察胞外体对淋巴细胞增殖的影响。结果:从2×107个细胞可获得20mL纯化的胞外体,测得其总蛋白浓度为(1.84±0.01)g/L,经电镜鉴定MCF-7细胞来源的胞外体直径为55～70nm,为有膜包裹的囊泡状结构,大小较为均一,形态特征与观察到的多泡体的内部囊泡的大小和形态是一致的。MTT试验显示它能够抑制IL-2诱导的淋巴细胞增殖,并且随剂量增加抑制作用增强。结论:肿瘤细胞来源的胞外体可通过细胞培养上清分步离心浓缩纯化得到,其不仅具有胞外体的形态学特征,同时可对IL-2诱导的淋巴细胞增殖产生剂量依赖性的抑制作用。Objective：To separate and identify the exosomes derived from MCF-7 tumor cells,and observe their function on IL-2 induced lymphocyte proliferation.Methods：The exosomes were separated by serial centrifugation,concentration and density gradient centrifugation, and then identified by transmission electron microscope.The effects of exosomes on IL-2 in- duced proliferation were studied by MTT method.Results：Total 20 mL exosomes from 2×107 MCF-7 cells with concentration of protein were （1.84±0.01） g/L. The diameters of these purified bi-layer membrane exosomes were 55-70 nm, which were consistent with the internal vesicles in multi-vesicular endosomes.The result of MTT test showed that proliferation of donor pe- ripheral blood lymphocytes was inhibited by tumor exosomes at a dose-dependent manner in response to IL-2.Conclusion：It is feasible to separate high-purity exosomes from tumor cell culture medium supernatant by serial centrifugation and concen- trate method. These tumor derived exosomes have inhibitory effect on IL-2 induced proliferation.

关 键 词：细胞系 肿瘤 乳腺肿瘤 免疫耐受 细胞分离 肿瘤细胞 培养的 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R284.1[医药卫生—基础医学]