外泌体搭载平阳霉素抑制乳腺癌肿瘤干细胞活力和侵袭的研究

Study of pingyangmycin-loaded exosomes inhibiting the proliferation and invasion of breast cancer stem cells

目的探索乳腺癌细胞来源的外泌体搭载平阳霉素对乳腺癌肿瘤干细胞恶性生物学行为的影响。方法用流式细胞术从MDA-MB-231乳腺癌细胞中筛选出CD133阳性乳腺癌肿瘤干细胞,用差速离心法分离外泌体,并将15,30和60 mg·mL^(-1)平阳霉素搭载到外泌体。将肿瘤干细胞分为空白组、对照组和低、中、高剂量实验组。空白组置于DMEM培养基进行常规培养;对照组用4 Gy X射线进行照射1次,照射时间20 min,而后置于DMEM培养基进行常规培养;低、中、高剂量实验组置于含300μg·mL^(-1)外泌体(载药量分别为15,30和60 mg·mL^(-1)平阳霉素)的DMEM培养基进行常规培养。用Transwell法检测细胞侵袭能力,用肿瘤球形成实验检测乳腺癌细胞体外肿瘤形成能力。结果低、高剂量实验组和对照组、空白组的Transwell穿越细胞数分别为(54.32±4.67),(38.75±4.33),(68.67±4.67)和(107.65±8.37)个,肿瘤球个数分别为(118.29±13.21),(46.27±9.93),(132.01±10.15)和(153.25±16.54)个,肿瘤球体积分别为(124.05±19.56),(64.68±17.72),(156.64±34.42)和(215.25±27.64)μm;。高剂量实验组的上述指标与对照组比较,差异均有统计学意义(均P<0.05)。结论外泌体搭载平阳霉素较单用平阳霉素更能显著抑制乳腺癌肿瘤干细胞的增殖和侵袭能力。

Objective To explore the effect of exosomes derived from breast cancer cells with pingyangmycin on the malignant biological behavior of breast cancer stem cells.Methods Flow cytometry was used to screen CD133-positive breast cancer stem cells from MDA-MB-231 cells,the exosomes were separated by differential centrifugation,and15,30 and 60 mg·m L^(-1)pingyangmycin were carried to exosomes.The tumor stem cells were divided into blank group,control group and experimental-L,-M and-H groups.The blank group was given DMEM medium for routine culture.The control group was irradiated with4 Gy X-ray once for 20 min,and then DMEM medium was given for routine culture.The experimental-L,-M,-H groups were routinely cultured in DMEM medium containing 300μg·m L^(-1)exosomes,the drug-loading amounts were 15,30 and 60 mg·m L^(-1)pingyangmycin.Transwell was used to detect cell invasion ability.The tumor formation capacity of breast cancer cells was determined by tumor sphere formation assay in vitro.Results The numbers of Transwell crossing cells in the experimental-L group,experimental-H group,control group and blank group were(54.32±4.67),(38.75±4.33),(68.67±4.67)and(107.65±8.37),the numbers of tumor sphere were(118.29±13.21),(46.27±9.93),(132.01±10.15)and(153.25±16.54),the tumor sphere volume was(124.05±19.56),(64.68±17.72),(156.64±34.42)and(215.25±27.64)μm;,respectively.The differences were statistically significant between experimental-H group and control group(all P<0.05).Conclusion Exosomes carrying pingyangmycin can significantly inhibit the proliferation and invasion of breast cancer stem cells.