高转移胃癌细胞株的建立及其肿瘤干细胞的生物学特征  

Establishment of a gastric cancer cell line with high metastasis and biological characteristics of cancer stem cell of the cell line

作　　者：遇珑[1] 舒雄[1] 刘辉琦[1] 刘军[1] 孙力超[1] 孙立新[1] 杨治华[1] 冉宇靓[1] 

机构地区：[1]国家癌症中心/中国医学科学院北京协和医学院,肿瘤医院,细胞及分子生物学实验室,北京100021

出　　处：《中国肿瘤生物治疗杂志》2016年第4期486-491,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金(No.31371445);国家高技术研究发展计划(863计划)(No.2014AA020537)

摘　　要：目的:建立高转移胃癌细胞株并研究其肿瘤干细胞生物学特征。方法:将胃癌细胞株SNU-5接种至裸鼠皮下成瘤后,取肺部转移灶,通过机械分离法并反复接种裸鼠,获取细胞株SNU-5-V12,采用无血清悬浮培养及PKH26染色确定SNU-5-V12细胞中存在肿瘤干细胞。流式细胞术分析SNU-5和SNU-5-V12细胞中CD44肿瘤干细胞标志物表达情况,分选SNU-5和SNU-5-V12细胞中CD44^+细胞并进行体外生物学特征研究及裸鼠致瘤实验。结果:建立高转移SNU-5-V12细胞株,SNU-5-V12细胞无血清悬浮培养11 d后形成的细胞球体中存在单个PKH26阳性细胞。流式分析显示高转移SNU-5-V12细胞中干细胞标志物CD44比例显著高于SNU-5细胞[(72.9±1.5)%vs(8.96±1.2)%],且SNU-5-V12的CD44^+细胞比SNU-5的CD44^+细胞具有更强的自我更新能力[成球率(27.8±1.7)%vs(20.4±1.0)%,P<0.01],转移细胞数增加1.64倍[(329.5±7.5)vs(200±2.0)个,P<0.01],耐药能力也显著增强[IC50:(0.286 vs 0.196)μg/ml,P<0.01],裸鼠皮下接种2×102个CD44^+SNU-5-V12细胞2个月2/6裸鼠可致瘤,而CD44^+SNU-5细胞需要接种2×104个细胞2个月时仅1/6裸鼠致瘤。结论:获得高转移胃癌细胞株SNU-5-V12,其CD44^+细胞体内外功能显著增强,为胃癌干细胞的靶向治疗提供有价值的细胞模型。Objective： To establish a gastric cancer cell line with high metastasis and explore biological characteristics of cancer stem cell for the cell line. Methods： The gastric cancer SNU-5 line cells were subcutaneously injected into nude mice. After a xenotransplanted tumor was formed,pulmonary metastasis of the tumor was taken out and repeatedly injected into nude mice by mechanical separation method,as well as the SNU-5-V12 cell line was achieved. Serum free suspension culture and PKH26 staining were used to confirm that there are cancer stem cells in the SNU-5-V12 cells. Expressions of cancer stem cell marker CD44 in the SUN-5 and the SUN-5-V12 cell lines were analyzed with flow cytometry assay. CD44^＋ cells in the SNU-5 and the SNU-5-V12 cell lines were separated and research in vitro of biological characteristics and tumorigenic experiments in nude mice were performed with the CD44^＋ cells. Results： The SNU-5-V12 cell line with high metastasis was established. There was a single PKH26 positive cell in spheroids formed by the SNU-5-V12 cells after serum free suspension culture for 11 days. Analysis of flow cytometry shown that ratio of cancer stem cell marker CD44 in SNU-5-V12 cell with high metastasis was significantly higher than that in the SNU-5 cell（ [72. 9 ± 1. 5]% vs [8. 96 ± 1. 2]%）,and self-renewal capacity of the CD44^＋ cells in the SNU-5-V12 cells was obviously stronger than that in the SNU-5 cells（ forming spheroid rate： [27. 8 ± 1. 7]% vs [20. 4 ± 1. 0]%,P〈0. 01）,as well as invasion ability of the SNU-5-V12 cells increased 1. 6times（ [329. 5 ± 7. 5]vs [200 ± 2. 0],P〈0. 01）,its drug resistant ability also enhanced significantly（ IC50： [0. 286 vs 0.196]μg / ml,P〈0. 01）. At two months of the injection,third one nude mice（ 2 /6） that injected with 2 × 102 the SNU-5-V12 cells formed xenotransplanted tumor,but only minority nude mice（ 1 /6） that injected with 2 × 104 the SNU-5 cells formed xenotransplanted tumor. Conclusion： A gastric cancer the SNU-

关 键 词：胃癌 肿瘤干细胞 转移 CD44 

分 类 号：R735.2[医药卫生—肿瘤] R730.2[医药卫生—临床医学]