抗肿瘤干细胞药物体外活性评价方法

Method Established for Evaluation of Anti-Cancer Stem Cell Drug Activity in vitro

目的以乳腺癌MCF-7细胞和盐霉素钠为例,建立体外抗癌干细胞药物活性评价的简单方法。方法无血清、含有生长因子的DMEM/F12培养基培养MCF-7细胞,观察细胞的生长及体外干细胞球形成能力;流式细胞仪检测CD44+/CD24-细胞含量;将无血清培养的、富集的MCF-7干细胞以不同的数量接种到Nu/Nu裸鼠皮下,观察并检验致瘤能力;CCK-8法检测药物对悬浮乳状球样细胞生长的作用。结果用无血清、含有生长因子的DMEM/F12培养基培养的MCF-7细胞,增殖缓慢,呈悬浮乳状球样;流式细胞仪检测CD44+/CD24-细胞含量,10%胎牛血清的RPMI1640培养的MCF-7干细胞为(12.8±0.6)%,无血清培养的MCF-7干细胞为(97.1±2.4)%;裸鼠接种无血清培养的、富集的MCF-7干细胞约100个细胞即可见肿瘤生成。盐霉素钠对无血清培养条件下的富含癌干细胞的MCF-7细胞有较高的毒性。结论所获得的CD44+/CD24-细胞具有乳腺癌干细胞特性,且超低黏附培养板和无血清环境可以促进悬浮球状细胞团的形成,CCK-8法是一种简便的体外评价抗癌干细胞药物的检测方法。

Objective To establish an simple in vitro anticancer drug activity evaluation method against cancer stem cells by creating a model based on breast cancer MCF-7 cell line-derived cancer stem cells and salino- mycin sodium exposure. Methods The MCF-7 cell growth and sphere-forming ability of stem cells in a serum-free DMEM/F12 medium were observed. The CD44 ＋/CD24- cell marker ratio of MCF-7 cells cultured at different con- ditions was detected through flow cytometry. We implanted serum-free cultured and enriched MCF-7 stem cells of different numbers subcutaneously in the flank of Nu/Nu nude mice, observed and checked the ability of tumorige- nicity, and evaluated the inhibitive effect of salinomycin sodium on suspended sphere-like cancer stem cells with the CCK-8 method. Results MCF-7 cells cultured in serum-free medium grew more slowly and formed suspended spheres. MCF-7 cells cultured in RPMI1640 contained （12.8 ±0.6）% CD44 ＋/CD24- cell marker while the ratio in serum-free medium was （97.1 ± 2.4 ） %. In the subcutaneous transplantation mouse model, about 100 MCF-7 stem cells cultured and enriched in serum-free medium led to a solid tumor formation. Salinomycin sodium was more toxic against MCF-7 stem cells cultured in serum-free medium. Conclusion CD44 ＋/CD24- cells possess breast cancer stem cell properties. The environment of ultra-low adhesion plates and serum-free medium can promote and maintain the formation of sphere-like cancer stem cells. CCK-8 method is a well-established activity evaluation method for targeting to cancer stem cell drugs in vitro.