LoVo细胞系中结肠癌干细胞样细胞的分离、培养及鉴定  

Isolation,culture and identification of colon cancer stem-like cells from LoVo cell line

作　　者：张洪也[1] 程勇[1] 胡祥[1] 吕原[1] 武乃金[1] 

机构地区：[1]重庆医科大学附属第一医院胃肠外科,重庆400016

出　　处：《中国肿瘤生物治疗杂志》2010年第2期149-154,共6页Chinese Journal of Cancer Biotherapy

基　　金：重庆市自然科学基金资助项目(No.2008BB5230)~~

摘　　要：目的:从结肠癌LoVo细胞系中分离、鉴定具有CD44+/EPCAMhigh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+/EPCAMhigh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+/EPCAMhigh、EPCAMlow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+/EPCAMhigh细胞中CD44/EPCAM的表达。结果:LoVo细胞中有17.4%的CD44+/EPCAMhigh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+/EPCAMhigh细胞增殖能力高于EPCAMlow细胞及未分选LoVo细胞,且细胞周期多集中在G0/G1期。以500个CD44+/EPCAMhigh细胞接种裸鼠成瘤率为90%(9/10),而1×104个EPCAMlow细胞成瘤率为0(0/10)。小鼠移植瘤中次代CD44+/EPCAMhigh细胞仍能少量表达CD44和EPCAM。结论:LoVo细胞中存在CD44+/EPCAMhigh结肠癌干细胞样细胞,CD44+/EPCAMhigh可用于结肠癌肿瘤干细胞的深入研究。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^＋/EPCAMhigh cells were observed. The proliferations and cell cycle distributions of CD44 ^＋/EPCAM^high, EPCAM^low, and un-sorted LoVo cells were estimated by MTF 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 immunofluorescenee 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^ ＋/EPCAMhigh cells was mostly in G0/G1 phase. Tumor formation rate or500 CD44^＋/EPCAM^high cells was 90% （9/10） in nude mice, with 1 × 10^4 EPCAM^low cells being 0% （0/10）. Moreover, expressions of CD44 and EPCAM in the second passage of CD44^ ＋/EPCAMhigh 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.

关 键 词：结肠癌 LOVO细胞株 肿瘤干细胞 干细胞样细胞 CD44/EPCAM 

分 类 号：R285.5[医药卫生—中药学]