悬浮培养结肠癌细胞球具有上皮间质转化特征  

Spheroid cells derived from human colon cancer cells exhibit epithelial to mesenchymal transitioncharacteristics

作　　者：冷政伟[1] 尹碧辉[2] 李勇[1] 谭俊[1] 岳中屹[1] 陈锦皇[1] 奚海林[1] 夏清华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院普外科,武汉430022 [2]仙桃市第一人民医院普外科

出　　处：《中华实验外科杂志》2013年第3期464-467,共4页Chinese Journal of Experimental Surgery

摘　　要：目的无血清悬浮培养人结肠癌DLD一1细胞形成细胞球，检测其肿瘤干细胞标志表达变化，并探讨球细胞发生上皮间质转化与肿瘤干细胞及肿瘤恶性行为之间的联系。方法在添加生长因子的无血清培养基中培养DLD一1细胞形成球细胞，运用Transwell实验检测细胞的迁移、侵袭能力变化；裸鼠皮下成瘤和软琼脂克隆形成实验分别检测体内外成瘤能力变化；流式细胞术检测细胞中CDl33表达变化；实时定量聚合酶链反应（Real—timePCR），Westernblot检测CDl33、CDl66、富含亮氨酸重复序列G一蛋白偶联受体5（Lgr5）及上皮问质转化关键基因表达变化。结果球细胞中CD133^＋细胞明显增加[（0．90±0．03）％LL（2．70±0．35）％，P〈0．05]；高表达肿瘤干细胞标志CDl33、CDl66、Lgr5和间质分子波形蛋白（Vimentin）及Snail而低表达上皮细胞标志ZO一1（P〈0．05）。球细胞的转移、侵袭、体内外成瘤能力明显增加（P〈0．05）。结论虽然黏钙蛋白（E—cadherin）表达明显升高，但是结合体内外实验结果，我们认为：E—cadherin可能不是介导细胞恶性行为的独立因素，悬浮培养形成的结肠癌球细胞已发生上皮间质转化，表现出肿瘤干细胞的特征。Objective To investigate the similarities between cancer stem cells and epithelial mes- enchymal transition （EMT） cells in spheroid cells. Methods Generate cancer spheroid cells from colon cancer cell line DLD-1 in serum free medium （SFM） and to investigate the expression of colon cancer stem cells markers such as CD133, CD166,1eucine-rich-repeat-containing G-protein-coupled receptor 5 （ Lgr5 ） and the numbers of CD133^＋ cells. In addition, the capacity of migration,invasion and tumorigenicity in vivo and in vitro were confirmed by transwell assay, colony formation assay and mouse xenograft model respec- tively. Moreover, the expression of EMT-related genes was detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting. Results The spheroid cells exhibit higher migra- tion,invasion,colony-formation and tumorigenicity properties. The CD133^＋ subpopulation was increased in spheroid cells. Moreover, the colon cancer stem cell markers such as CD133, CD166, Lgr5 and mesenchymal genes such as Vimentin and Snail were increased while ZO-1 decreased in spheroid cells. Surprisingly, E- cadherin-the key mediator of EMT was higher expressed too. Conclusion Although the E-cadherin was in- creased expression, considered the experiments in vitro and in vivo, we thought that E-cadherin may be not the sole cause of the malignant files in spheroid cells. Moreover, the spheroid cells exhibit cancer stem cells nrooerties and EMT Characteristics.

关 键 词：结肠癌 肿瘤干细胞 上皮间质转化 

分 类 号：R735[医药卫生—肿瘤]