人食管鳞癌细胞系EC109中肿瘤干细胞的分离(富集)与鉴定

目的 运用无血清成球培养法从人食管鳞癌细胞系EC109中分离/富集肿瘤干细胞,并对其“干性”特性进行鉴定.方法 将EC109细胞接种至添加1X B27的干细胞培养基(DMEM/F12 1∶1,无血清),于poly-Hema包被的培养皿或低黏附培养板中培养,获取细胞球.采用实时定量PCR方法检测其干性相关转录因子的基因表达,连续传代和平板克隆形成实验评价其自我更新能力,免疫荧光细胞化学法检测其分化前后干性相关转录因子和分化标志物的表达变化,以裸鼠移植瘤实验检测其体内成瘤能力.结果 培养5d能获得EC109细胞的细胞球.细胞球细胞高表达Oct4等干性相关转录因子,具有自我更新能力、分化潜能.裸鼠体

Isolation(enrichment) and characterization of cancer stem cells in esophageal sequamous cell carcinoma cell line EC109

Objective To isolate/enrich and characterize the cancer stem cells in esophageal sequamous cell carcinoma cell llne EC109 using method of serum-free formation. Methods EC109 cells were cultivated in poly- Hema coated dish or commercial low adhesion plate with serum-free stem cell medium （DMEM/F12 1： 1） supplemented with 1X B27 and the cancer stem cells reforming into floating spheres were isolated. The expression of sternness relative transcriptional factors of the spheres was measured by qRT-PCR. Continuous passage and plate clone formation was employed to estimate the self-renewal capacity. Immunofluorescence cytochemistry was used to detect the changes of sternness relative transcriptional factors and differentiation markers expressed in spheres before and after differentiation. To determine tumorigenesis in vivo, Xenograft assay in nude mice was performed respectively. Results Tumor spheres were usually generated at day 5. Compared with monolayer adherent cells, isolated tumor sphere cells highly expressed sternness relative transcriptional factors,and possessed self-renewal and differentiation potential, and exhibited enhanced tumorigenicity in immuno-deficient mice. Also, the size of sphere cell-derived xenograft tumors was larger than monolayer adherent cell-derived tumors. Conclusion Tumor spheres generated from EC109 cells under serum-free stem cell medium and floating culture enriched cancer stem cells.