外泌体在体外对乳腺癌细胞耐药信息传递的作用

The role of exosomes in drug resistance information transmission of breast cancer in vitro

目的探讨外泌体(exosomes,EXOs)在乳腺癌细胞间耐药信息的传递作用。方法选用乳腺癌敏感细胞株(MCF-7)及其阿霉素耐药株(MCF-7/ADR)为模型,用超速离心法提取MCF-7/ADR外泌体(ADR/EXO);用透射电子显微镜观察鉴定细胞外泌体形态;运用倒置荧光显微镜观察ADR/EXO与MCF-7细胞的相互作用过程;运用激光共聚焦显微镜观察MCF-7与MCF-7/ADR在Transwell小室共培养60h后细胞内阿霉素药物浓度变化;运用CCK-8法检测ADR/EXO与MCF-7细胞共培养后对阿霉素的IC50的影响;运用RT-PCR检测MCF-7与ADR/EXO共培养后MDR1基因表达水平的变化;运用Western blot法检测MCF-7与ADR/EXO共培养12h和72h后P-gp表达水平的变化。结果透射电子显微镜下,外泌体呈现典型的圆形或椭圆形杯口状结构,直径为40~100nm;用PKH67标记ADR/EXO显示,ADR/EXO是可以被MCF-7细胞摄取;与ADR/EXO共培养后敏感细胞株MCF-7对阿霉素的IC50耐药性升高2.62倍,差异有统计学意义(P<0.05);经过与MCF-7/ADR在Transwell小室中共培养60h后的MCF-7细胞,胞内可见阿霉素聚集减少,核区分布减少,荧光强度减弱;MCF-7+ADR/EXO组MDR1的mRNA表达量明显增加,是MCF-7组的8.69倍,差异有统计学意义(P<0.05);与MCF-7组相比,共培养12h的MCF-7+ADR/EXO组P-gp相对表达量升高4.64倍,差异有统计学意义(P<0.05);与MCF-7组相比,共培养72h的MCF-7+ADR/EXO组P-gp相对表达量升高3.37倍,差异有统计学意义(P<0.05)。结论乳腺癌外泌体可能具有传递耐药信息的作用,耐药细胞外泌体能够使敏感细胞获得一定的耐药性,耐药细胞外泌体向敏感细胞传递P-gp,不仅可以通过蛋白形式进行传递,而且可以通过mRNA的形式进行传递。

Objective To study the function of exosomes（EXOs）in transmitting drug resistance information between breast cancer cells.Methods Breast cancer cell lines MCF-7 and MCF-7/ADR were selected as experimental cells.Exosomes derived from MCF-7/ADR were isolated from the supernatants by ultracentrifugation.The morphology of isolated exosomes was observed by transmission electron microscopy（TEM）and the interaction between ADR/EXO and MCF-7 cells was observed under the inverted fluorescence microscope.Cell uptake of adriamycin was observed under the laser confocal microscope after 60 h of coculture of MCF-7 and ADR/EXO.CCK-8 assay was used to detect the changes of adriamycin IC50 value of MCF-7 after co-cultured with ADR/EXO cells.The effect of ADR/EXO on MCF-7 MDR1 mRNA was determined by RT-PCR.Western blot was used to examine P-gp expression at 12 h and 72 h after co-culture.Results Under the transmission electron microscope,the isolated EXOs were round or oval in shape with a diameter between 40 and 100 nm.ADR/EXO labeled with PKH67 could be uptaken by MCF-7 cells.After incubation with ADR/EXO,adriamycin IC50 of sensitive cell line MCF-7 increased by 2.62 times,with a significant difference（P〈0.05）.After MCF-7 cells were co-cultured with MCF-7/ADR cells in the Transwell model for 60 h,adriamycin fluorescence intensity became weaker than that of MCF-7.The expression of MDR1 mRNA was significantly higher in MCF-7＋ADR/EXO group than in MCF-7group（P〈0.05）.Compared with that in MCF-7 groups,the relative expression of P-gp co-cultured with ADR/EXO for 12 h significantly increased by 4.64 times（P〈0.05）;co-cultured with ADR/EXO for 72 h,the relative expression increased by 3.37 times,with a significant difference（P〈0.05）.Conclusion Our results suggest that the EXOs from breast cancer cells can transmit the drug resistance message,sensitive cells can acquire drug resistance from the EXOs of drug-resistant cells.P-gp can be transferred between breast cancer cells,not only in the form of protein but also in the form of mRNA.