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- 2017
外泌体在体外对乳腺癌细胞耐药信息传递的作用
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Abstract:
摘要：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。
ABSTRACT: 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 60h 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 12h and 72h after co-culture. Results Under the transmission electron microscope, the isolated EXOs were round or oval in shape with a diameter between 40 and 100nm. 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 60h, 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-7 group (P<0.05). Compared with that in MCF-7 groups, the relative expression of P-gp co-cultured with ADR/EXO for 12h significantly increased by 4.64 times (P<0.05); co-cultured with ADR/EXO for 72h, the relative
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