摘 要 目的：从结肠癌LoVo细胞系中分离、鉴定具有CD44+/EPCAM high特异表型的结肠癌干细胞样细胞，观察其生物学行为，证实该细胞系中结肠癌干细胞样细胞的存在。方法：从普通血清培养的LoVo细胞系中以流式细胞仪分选具有CD44+/EPCAM high表型的细胞，接种于添加生长因子的无血清培养基中，观察其增殖过程，继而诱导分化。MTT法、流式细胞术检测CD44+/EPCAM high、EPCAM low和未分选LoVo细胞的增殖能力及细胞周期分布。3种细胞接种裸鼠，比较不同细胞的成瘤率；免疫荧光技术检测小鼠次代CD44+/EPCAM high细胞中 CD44/EPCAM的表达。结果：LoVo细胞中有17.4%的CD44+/EPCAM high细胞，并能在添加生长因子的无血清培养基中呈细胞球样生长，且可连续传代；在血清的诱导下，呈贴壁分化生长，其形态与未分选LoVo细胞无差别。CD44+/EPCAM high细胞增殖能力高于EPCAM low细胞及未分选LoVo细胞，且细胞周期多集中在G0/G1期。以500个CD44+/EPCAM high细胞接种裸鼠成瘤率为90%（9/10），而1×104个EPCAM low细胞成瘤率为0（0/10)。小鼠移植瘤中次代CD44+/EPCAM high细胞仍能少量表达CD44和EPCAM。结论：LoVo细胞中存在CD44+/EPCAM high结肠癌干细胞样细胞，CD44+/EPCAM high可用于结肠癌肿瘤干细胞的深入研究。

Abstract Objective:To isolate, culture and identify the colon cancer stem-like cells with CD44+/EPCAM high phenotype from LoVo cell line, and to observe their biological behaviors and verify the existence of tumor stem cells in LoVo cell line. Methods: CD44+/EPCAM high cells were sorted from LoVo cells cultured in common-serum medium by flow cytometry, and the resultants were further cultured in serum-free medium (SFM) supplemented with growth factors. The proliferation and differentiation of CD44+/EPCAM high cells were observed. The proliferations and cell cycle distributions of CD44+/EPCAM high, EPCAM low, and un-sorted LoVo cells were estimated by MTT and flow cytometry, respectively. Nude mice were implanted with the above 3 different cells, and tumor formation rates of different groups were analyzed. Expressions of CD44 and EPCAM in the second passage of CD44+/EPCAM high cells were determined by immunofluorescence staining. Results: We found that 17.4% of LoVo cells were CD44+/EPCAM high cells, which could steadily proliferate and assemble into tumor cell spheres in SFM supplemented with growth factors. CD44+/EPCAM high cells could differentiate into adherent cells by serum, similar to LoVo cells. The proliferation capacity of CD44+/EPCAM high cells was higher than those of EPCAM low and LoVo cells, and the cell cycle of CD44+/EPCAM high cells was mostly in G0/G1 phase. Tumor formation rate of 500 CD44+/EPCAM high cells was 90% (9/10) in nude mice, with 1×104 EPCAM low cells being 0% (0/10). Moreover, expressions of CD44 and EPCAM in the second passage of CD44+/EPCAM high cells could still be detected. Conclusion: Colon cancer cell line LoVo contains CD44+/EPCAM high stem-like cells, and CD44+/EPCAM high cells can be used in further research of colon cancer stem cells.

重庆市自然科学基金资助项目（No. 2008BB5230）