目的： 分离并鉴定人胃癌细胞系SNU-5中的肿瘤干细胞，探讨人胃癌CD90+干细胞对胃癌转移和预后的影响。 方法： 无血清悬浮培养及PKH26染色确定SNU-5细胞系中是否存在肿瘤干细胞，流式细胞术分析SNU-5亲本及球体细胞中肿瘤干细胞标志物的表达，分选CD90+SNU-5细胞并进行体外生物学特征研究及SCID鼠致瘤实验。收集肿瘤医院腹部外科95例胃癌患者肿瘤病理标本，免疫组化方法检测胃癌组织中CD90的表达。 结果： SNU-5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。无血清悬浮培养可将CD90+SNU-5细胞富集6.1倍，且CD90可在球体细胞中与标示干细胞的PKH26共染。CD90+SNU-5细胞较CD90- SNU-5细胞和亲本SNU-5细胞具有更高的自我更新能力\[成球率（7.7±1.1）% vs (1.3±0.4)%、(1.8±0.3)%，均P<0.01\]和侵袭能力\[侵袭细胞数(283.3±30.2) vs (48.0±7.5)、(156.7±72)个，均P<0.01\]。CD90+SNU-5细胞在重症联合免疫缺陷（severe combined immune deficency，SCID）小鼠皮下接种2×102个细胞6周即可致瘤（1/6），而接种2×104个CD90- SNU-5细胞10周才能致瘤（1/6）。95例胃癌患者组织中CD90的表达与胃癌的远处转移显著相关（P<0.01），且CD90阳性胃癌患者的生存期明显短于CD90阴性的患者（P<0.01）。 结论： 人胃癌细胞系SNU-5中存在具有更强自我更新及侵袭能力的CD90+干细胞，人胃癌组织中CD90+干细胞数量与肿瘤的转移与患者生存期明显相关。

Objective:To isolate and identify the cancer stem cells from gastric cancer cell line SNU-5, and investigate the influence of CD90+ cancer stem cells on the metastasis and prognosis of gastric cancer. Methods: Non-adherent culture and PKH26 staining was used to determine whether there were cancer stem cells in human gastric cancer cell line SNU-5; Flow cytometry analysis was performed for identification of positive cells with stem cell markers within parent SNU-5 cells and sphere cells; CD90+ SNU-5 cells were sorted to identify its biological characteristics in vivo and tumorigenicity in vitro. Ninety-five cases with gastric cancer selected from cancer Hospital, immunohistochemistry was applied to detect CD90 expression in human gastric cancer. Results: The single PKH26 positive cell was observed in spheroids after SNU-5 cells were cultured for 11 days and CD90 could be co-stained with PKH26 in the spheroid cells, a dye indicating the characterization of stem cells. Serum sphere culture can enrich CD90+ SNU-5 cells by 6.1 times. The sorted CD90+ SNU-5 cells presented higher self-renewal ability (sphere formation rate: \[7.7±1.1\]% vs \[1.3±0.4\]%, \[18±03\]%，P<0.01), higher invasion (the number of invasive cells \[283.3±30.2\] vs \[48.0±7.5\], \[156.7±7.2\], P<0.01)than CD90- SUN-5 cells and parent SNU-5 cells. Only 2×102 CD90+ SNU-5 cells can form tumors at 6 weeks (4/6) in severe combined immune deficency (SCID) mice, whereas at least 2×104 CD90- SNU-5 cells were needed to form tumors at 10 weeks (1/6). Immunohistochemical results showed that CD90 expression was significantly associated with distant metastasis of gastric cancer (P<0.01). The survival rate of CD90 positive gastric cancer patients is obviously lower than that of CD90 negative patients (P<0.01). Conclusion: CD90+ cancer stem cells in huamn gastric cancer cell line SNU-5 show increased self-renewal and invasion abilitities, which are related to metastasis and prognosis of gastric cancer.

国家科技重大专项课题资助（No. 2011ZX09102-010-02）