CAJ | 학술논문

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,Western blot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 nm.Exosomes仅表达Exosomes标志性蛋白Tsg101,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-7/S细胞共培养72 h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24 h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48 h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着耐药性传递的现象,Exosomes可能是耐药信息传递的转运载体.
목적 탐토유선암다서자삼순(DOC)내약세포(MCF-7/DOC)화아매소(ADM)내약세포(MCF-7/ADM)대Exosomes적분비공능급기재내약성전체중적작용.방법 채용초속분급리심적방법,종유선암내약세포배양액상청중제취Exosomes,투사전경하관찰기형태특정,Western blot법검측유선암내약세포화Exosomes중표지단백적표체정황.이용중조만병독재체구건은정표체록색형광단백(GFP)적유선암친본민감세포계GFP-MCF-7/S,통과세포간내약실험화Exosomes전체내약성실험관찰내약성전체현상.결과 여MCF-7/S세포상동,MCF-7/DOC화MCF-7/ADM세포야분비Exosomes,균위원형혹타원형적막성낭포,직경위30 ～ 100 nm.Exosomes부표체Exosomes표지성단백Tsg101,불표체내질망표지단백Calnexin.MCF-7/S、MCF-7/DOC혹MCF-7/ADM세포여GFP-MCF-7/S세포공배양72 h후,형광현미경하관찰각조세포적형광표체정황무명현차별.단재가입화료약물DOC혹ADM처리24 h후,MCF-7/DOC+ GFP-MCF-7/S조세포적형광세포평균존활솔위65.5％,명현고우MCF-7/S+ GFP-MCF-7/S조세포(25.5％,P＜0.001);MCF-7/ADM+ GFP-MCF-7/S조세포적형광세포평균존활솔위53.6％,역명현고우MCF-7/S+ GFP-MCF-7/S조세포(25.4％,P＜0.001).MCF-7/S、MCF-7/DOC혹MCF-7/ADM세포래원적Exosomes여GFP-MCF-7/S세포공배양48 h후,형광현미경하관찰각조세포적형광표체정황무명현차별.단재가입화료약물DOC혹ADM처리24h후,MCF-7/DOC래원적Exosomes+ GFP-MCF-7/S조세포적형광세포평균존활솔위59.9％,명현고우MCF-7/S래원적Exosomes+ GFP-MCF-7/S조세포(32.4％,P＜0.001);MCF-7/ADM래원적Exosomes+GFP-MCF-7/S조세포적형광세포평균존활솔위58.3％,역명현고우MCF-7/S래원적Exosomes+ GFP-MCF-7/S조세포(27.2％,P＜0.001).결론 유선암세포간존재착내약성전체적현상,Exosomes가능시내약신식전체적전운재체.
Objective To explore whether docetaxel-resistant cells (MCF-7/Doc) and doxorubicinresistant 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 supernatants of MCF-7/Doc and MCF-7/ ADM cells by fractionation ultracentrifugation,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,Tsg101,did not express the endoplasmic reticulum marker calnexin.After MGF-7/S,MCF-7/DOC and MCF-7/ADM cells 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 for24 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 survival rate of fluorescence-labeled cells compared with the exosomes extracted from MCF-7/S + GFP-MCF-7/S group (59.9％ vs.32.4％,P＜0.001),and so did the exosomes extracted from the MCF-7/ADM) + GFP-MCF-7/S group (58.3％ vs.27.2％,P ＜ 0.001).Conclusion Our results suggest that drugresistance can be transferred between breast cancer cells,and exosomes are probably the transporter of the drug resistance.