胆囊癌细胞株GBC-SD类肿瘤干细胞筛选的研究  

Preliminary study of screening and differentiating cancer stem-like cell colony in gallbladder carcinoma cell line GBC-SD

作　　者：殷保兵[1] 宗华杰[1] 马保金[1] 蔡端[1] 

机构地区：[1]复旦大学附属华山医院普外科,上海200040

出　　处：《中华实验外科杂志》2010年第7期857-859,共3页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家自然科学基金资助项目（30672056）

摘　　要：目的 筛选胆囊癌GBC-SD系中具有干细胞特性的细胞克隆.方法 在肿瘤干细胞培养基加入不同剂量的顺铂悬浮培养GBC-SD,将悬浮细胞收集后注射裸鼠,并持续瘤内注射顺铂,成瘤后取瘤内细胞继续原培养基培养,获得悬浮生长的克隆,分别测定其干细胞标志物（CD133、CD44、CD24）、耐药基因ABCG2细胞株MDR-1和转录因子OCT-4、Nanog的表达.结果 在肿瘤干细胞培养基和顺铂的筛选压力下,GBC-SD细胞能有效形成悬浮生长的克隆球,流式检测结果表明克隆球高表达CD133＋（97.6%）、CD44＋（77.9%）,低表达CD24＋（2.3%）,同时表达耐药基因ABCG2和MDR-1,定量聚合酶链反应（PCR）及免疫荧光方法检测发现,与普通胆囊癌细胞株GBC-SD比较,克隆球干细胞基因OCT-4、Nanog表达分别增强266、284倍.结论 顺铂结合无血清培养基悬浮培养法筛选GBC-SD,作为一种新的干细胞分选方法,可以分离出具有肿瘤千细胞特征的胆囊癌克隆球.Objective To screen and differentiate cancer stem-like cell colony in gallbladder carcinoma cell line CBC-SD. Methods CBC-SD cells was suspension cultured in non-serum medium containing cisplatin, sphere-forming cells of GBC-SD were isolated. These isolated cells were then transplanted into nude mice. After tumor formation,cisplatin was also injected into tumors repeatedly and then these tumor cells were isolated and purified and cultured in FBS-Free medium. FACS was used to detect expression of stem cell markers including CD133 ,CD44 and CD24. Immuno-cytochemistry and real time polymerase chain reaction （PCR） were used to examine drug-resistant gene such as ABCG2 and MDR-1. Also transcription factor such as OCT-4 and Nanog were detected using the same way. Results GBC-SD cells formed sphere colonies under the pressure of cisplatin and serum-free medium. FACS results showed 97. 6% of the cells expressed CD133,77. 9% of the cells expressed CD44,2. 3% of the cells expressed CD24. Real time PCR results showed these cells highly expressed stem cell markers Nanog and OCT-4,as well as drug-resistant gene ABCG2 and MDR-1. When these cells cultured in FBS contained medium, expression of stem cell markers such as OCT-4 and NANOG decreased, whereas expression of differentiation markers such as Mucland Vimentin increased. Conclusion Combined treatment with cisplatin and serum-free medium is a new way to isolate cancer stem-like cell colony from GBC-SD cell line.

关 键 词：GBC-SD 肿瘤干细胞 耐药 悬浮培养 

分 类 号：R735.8[医药卫生—肿瘤]