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\[IC50：(0.286 vs 0.196) μg/ml，P<0.01\]，裸鼠皮下接种2×102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2×104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 1.6 times (\[329.5±7.5\] vs \[200±20\], P<0.01）, its drug resistant ability also enhanced significantly (IC50: \[0.286 vs 0.196\]μg/ml, P<0.01). At two months of the injection, third one nude mice (2/6) that injected with 2×102 the SNU-5-V12 cells formed xenotransplanted tumor, but only minority nude mice (1/6) that injected with 2×104 the SNU-5 cells formed xenotransplanted tumor. Conclusion:A gastric cancer the SNU-5-V12 cell line with high metastasis was obtained. In vivo and in vitro functions of CD44+ cell in the SNU-5-V12 cell line significantly increased. It could provide a valuable cell model for target therapy of gastric cancer stem cell.

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)