Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China.
J Zhejiang Univ Sci B. 2011 Apr;12(4):256-63. doi: 10.1631/jzus.B1000303.
This paper aims to screen and identify sphere clone cells with characteristics similar to cancer stem cells in human gallbladder cancer cell line GBC-SD. GBC-SD cells were cultured in a serum-free culture medium with different concentrations of the chemotherapeutic drug cisplatin for generating sphere clones. The mRNA expressions of stem cell-related genes CD133, OCT-4, Nanog, and drug resistance genes ABCG2 and MDR-1 in sphere clones were detected by quantitative real-time polymerase chain reaction (PCR). Stem cell markers were also analyzed by flow cytometry and immunofluorescent staining. Different amounts of sphere clones were injected into nude mice to test their abilities to form tumors. Sphere clones were formed in serum-free culture medium containing cisplatin (30 μmol/L). Flow cytometry results demonstrated that the sphere clones expressed high levels of stem cell markers CD133(+) (97.6%) and CD44(+) (77.9%) and low levels of CD24(+) (2.3%). These clones also overexpressed the drug resistance genes ABCG2 and MDR-1. Quantitative real-time PCR showed that sphere clones expressed stem cell genes Nanog and OCT-4 284 and 266 times, respectively, more than those in the original GBC-SD cells. Immunofluorescent staining showed that sphere clones overexpressed OCT-4, Nanog, and SOX-2, and low expressed MUC1 and vimentin. Tumor formation experiments showed that 1×10(3) sphere clone cells could induce much larger tumors in nude mice than 1×10(5) GBC-SD cells. In conclusion, sphere clones of gallbladder cancer with stem cell-like characteristics can be obtained using suspension cultures of GBC-SD cells in serum-free culture medium containing cisplatin.
本文旨在筛选并鉴定人胆囊癌细胞系 GBC-SD 中具有类似癌症干细胞特征的球体克隆细胞。将 GBC-SD 细胞在含有不同浓度化疗药物顺铂的无血清培养基中培养，以生成球体克隆。通过实时定量聚合酶链反应（PCR）检测球体克隆中干细胞相关基因 CD133、OCT-4、Nanog 和耐药基因 ABCG2、MDR-1 的 mRNA 表达。通过流式细胞术和免疫荧光染色分析干细胞标志物。将不同数量的球体克隆注入裸鼠中，以测试其形成肿瘤的能力。在含有顺铂（30μmol/L）的无血清培养基中形成球体克隆。流式细胞术结果表明，球体克隆表达高水平的干细胞标志物 CD133(+)（97.6%）和 CD44(+)（77.9%），低水平的 CD24(+)（2.3%）。这些克隆还过度表达了耐药基因 ABCG2 和 MDR-1。实时定量 PCR 显示，球体克隆中 Nanog 和 OCT-4 的表达分别是原始 GBC-SD 细胞的 284 倍和 266 倍。免疫荧光染色显示，球体克隆过度表达 OCT-4、Nanog 和 SOX-2，低表达 MUC1 和波形蛋白。肿瘤形成实验表明，1×10(3)个球体克隆细胞比 1×10(5)个 GBC-SD 细胞能在裸鼠中诱导更大的肿瘤。综上所述，使用含顺铂的无血清培养基悬浮培养 GBC-SD 细胞可获得具有干细胞样特征的胆囊癌细胞球体克隆。
