乳腺癌耐药细胞来源的Exosomes的分离鉴定及其在耐药传递中的作用  

Extraction and identification of exosomes from drug-resistant breast cancer cells and their potential role in cell-to-cell drug-resistance transfer

作　　者：许金金[1] 李文静[1] 钟山亮[1] 李秀娟[2] 陈志远[2] 胡清[2] 唐金海[2] 赵建华[1] 

机构地区：[1]南京医科大学附属肿瘤医院江苏省肿瘤医院临床检验中心,210009 [2]南京医科大学附属肿瘤医院江苏省肿瘤医院普外科,210009

出　　处：《中华肿瘤杂志》2014年第3期165-170,共6页Chinese Journal of Oncology

基　　金：国家自然科学基金面上项目（81272470）

摘　　要：目的探讨乳腺癌多西紫杉醇（DOC）耐药细胞（MCF-7／DOC）和阿霉素（ADM）耐药细胞（MCF-7／ADM）对Exosomes的分泌功能及其在耐药性传递中的作用。方法采用超速分级离心的方法，从乳腺癌耐药细胞培养液上清中提取Exosomes，透射电镜下观察其形态特征，Westernblot法检测乳腺癌耐药细胞和Exosomes中标志蛋白的表达情况。利用重组慢病毒载体构建稳定表达绿色荧光蛋白（GFP）的乳腺癌亲本敏感细胞系GFP-MCF-7／S，通过细胞间耐药实验和Exosomes传递耐药性实验观察耐药性传递现象。结果与MCF-7／S细胞相同，MCF-7／DOC和MCF-7／ADM细胞也分泌Exosomes，均为圆形或椭圆形的膜性囊泡，直径为30～100／lm。Exosomes仅表达Exosomes标志性蛋白Tsgl01，不表达内质网标志蛋白Calnexin。MCF-7／S、MCF-7／DOC或MCF-7／ADM细胞与GFP—MCF一7／S细胞共培养72h后，荧光显微镜下观察各组细胞的荧光表达情况无明显差别。但在加人化疗药物DOC或ADM处理24h后，MCF-7／DOC＋GFP-MCF-7／S组细胞的荧光细胞平均存活率为65．5％，明显高于MCF-7／S＋GFP-MCF-7／S组细胞（25．5％，P〈0．001）；MCF-7／ADM＋GFP-MCF-7／S组细胞的荧光细胞平均存活率为53．6％，亦明显高于MCF-7／S＋GFP—MCF-7／S组细胞（25．4％，P〈0．001）。MCF-7／S、MCF-7／DOC或MCF-7／ADM细胞来源的Exosomes与GFP-MCF-7／S细胞共培养48h后，荧光显微镜下观察各组细胞的荧光表达情况无明显差别。但在加入化疗药物DOC或ADM处理24h后，MCF-7／DOC来源的Exosomes＋GFP—MCF-7／S组细胞的荧光细胞平均存活率为59．9％，明显高于MCF-7／S来源的Exosomes＋GFP—MCF-7／S组细胞（32．4％，P〈0．001）；MCF-7／ADM来源的Exosomes＋GFP-MCF-7／S组细胞的荧光细胞平均存活率为58．3％，亦明显高于MCF-7／S来源的Exosomes＋GFP—MCF-7／S组细胞（27．2％，P〈0．001）。结论乳腺癌细胞间存在�Objective To explore whether docetaxel-resistant cells （MCF-7/Doc） and doxombicin- resistant cells （MCF-7/ADM） can secrete Exosomes and their potential role in cell-cell drug-resistance transfer. Methods Exosomes were extracted from the cell culture supematants of MCF-7/Doc and MCF-7/ ADM cells by fractionation uhracentrifugation, and were identified by transmission electron microscopy and Western blot analysis. GFP-MCF-7/S, a breast cancer parental sensitive cell line stably expressing green fluorescent protein （ GFP）, was constructed by recombinant lentiviral vector with GFP. Then the resistance experiment of cells and the experiment of resistance transfer by exosomes were designed to observe the phenomenon of cell-to-cell drug-resistance transfer. Results Similar to the breast cancer parental sensitive cells （MCF-7/S）, the breast cancer resistant sublines could secrete exosomes, which exhibited round or elliptic shape ranging from 30 to 100 nm in diameter with intact membrane, and only expressed the protein marker of exosomes, Tsgl01, did not express the endoplasmic reticulum marker calnexin. After MCF-7/S, MCF-7/DOC and MCF-7/ADM ceils we cocultured with GFP-MCF-7/S cells for 72 h, there were no significant differences in the expression of fluorescence-labeled cells among the four groups. When treated by the drug ADM or DOC for＇24 hours, the MCF-7/DOC ＋ GFP-MCF-7/S group was in favor of a significant higher survival rate of fluorescence-labeled cells compared with the MCF-7/S ＋ GFP-MCF-7/S group （65.5% vs. 25.5%, P 〈0.001）, and so did the MCF-7/ADM ＋ GFP-MCF-7/S group （53.6% vs. 25.4%, P 〈 0.001 ）. The exosomes extracted from MCF-7/S, MCF-7/DOC and MCF-7/ADM cells were cultured with the GFP-MCF-7/S cells for 48 h. Among these groups, no significant differences in the expression of fluorescence-labeled cells were found. After treated by the drug ADM or DOC for 24 hours, the exosomes extracted from MCF-7/DOC ＋ GFP-MCF-7/S group was associated with a significant higher survi

关 键 词：细胞系 肿瘤 乳腺肿瘤 EXOSOMES 多西紫杉醇 阿霉素 抗药性 肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]