CAJ | 학술논문

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.
목적 사선담낭암GBC-SD계중구유간세포특성적세포극륭.방법 재종류간세포배양기가입불동제량적순박현부배양GBC-SD,장현부세포수집후주사라서,병지속류내주사순박,성류후취류내세포계속원배양기배양,획득현부생장적극륭,분별측정기간세포표지물(CD133、CD44、CD24)、내약기인ABCG2세포주MDR-1화전록인자OCT-4、Nanog적표체.결과 재종류간세포배양기화순박적사선압력하,GBC-SD세포능유효형성현부생장적극륭구,류식검측결과표명극륭구고표체CD133+(97.6%)、CD44+(77.9%),저표체CD24+(2.3%),동시표체내약기인ABCG2화MDR-1,정량취합매련반응(PCR)급면역형광방법검측발현,여보통담낭암세포주GBC-SD비교,극륭구간세포기인OCT-4、Nanog표체분별증강266、284배.결론 순박결합무혈청배양기현부배양법사선GBC-SD,작위일충신적간세포분선방법,가이분리출구유종류천세포특정적담낭암극륭구.
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.