摘要
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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第1期67-69,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(81172532)
教育部长江学者和创新团队计划资助项目(TRT0743)
关键词
乳腺癌干细胞
雄激素受体
Breast cancer stem cells
Androgen receptor