雄激素受体阳性乳腺癌干细胞的富集及其特性鉴定

Enrichment and identification of androgen receptor-positive breast cancer stem cells

目的 从雄激素受体阳性（AR＋）乳腺癌细胞株中分离出乳腺微球体（MSs）进行培养和鉴定.方法 将3种AR＋乳腺癌细胞株（MCF-7、MDA-MB-231及T47D）接种在添加生长因子的DMEM/F12培养基中,观察MSs的形成及生长；细胞免疫荧光检测MCF-7、MDA-MB-231及T47D中雄激素受体（AR）的表达；流式细胞术（FCM）检测贴壁细胞及MSs中CD44＋ CD24-/low细胞的含量；单克隆形成实验检测MSs细胞的自我更新能力.结果 乳腺癌细胞株在无血清培养基中呈悬浮生长,7-10d可形成MSs;细胞免疫荧光显示MCF-7、T47D中AR主要在细胞质中表达、MDA-MB-231中AR在细胞核及细胞质中均有表达；流式细胞术检测MCF-7、MDA-MB-231及T47D贴壁细胞中CD44＋ CD24-/low细胞含量分别为50.0％、86.4％、3.2％,悬浮细胞中CD44＋ CD24-/low细胞含量分为87.4％、98.8％、38.4％；克隆形成实验显示随着时间的推移形成的克隆球体积不断增大.结论 悬浮生长的MDA-MB-231及MCF-7细胞可作为研究AR阳性乳腺癌干细胞的良好模型.

Objective To isolate the mammospheres （MSs） from the androgen receptor positive（AR ＋） breast cancer cell lines for culture and identification to screen AR ＋ breast cancer stem cell model for further study.Methods The three types of AR ＋ breast cancer cell lines were seeded in culture medium DMEM/F12 supplemented with growth factors.The formation and growth of MSs were observed.Immunofluorescence was used to detect the expression of AR in MCF-7,MDA-MB-231 and T47D cell lines.Flow cytometry （FCM） was used to detect CD44 ＋ CD24-/low cell content in adherent cells and mammospheres.Clonogenic assay was used to detect the self-renew capcity of every single cell of the MSs.Results Breast cancer cell lines in serum-free medium showed suspension growth,and MSs formed during 7-10 days.Immunofluorescence showed MCF-7 and T47D cells expressed AR mainly in the cytoplasm,and MDA-MB-231 cells in both the nucleus and cytoplasm.FCM revealed that CD44＋ CD24-/low cell levels in MCF-7,MDA-MB-231 and T47D adherent cells were 50.0%,86.4% and 3.2%,and those in suspended cells were 87.4%,98.8% and 38.4%,respectively.Colony formation assay demonstrated that the volume of MSs was gradually increased with time.Conclusion Suspension growth of MDA-MB-231 and MCF-7 cells can be used as an optimal model for the study of AR-positive breast cancer stem cells.