CAJ | 학술논문

目的无血清悬浮培养人结肠癌DLD-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-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CD133表达变化；实时定量聚合酶链反应(Real-time PCR),Western blot检测CD133、CD166、富含亮氨酸重复序列G-蛋白偶联受体5(Lgr5)及上皮间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)％比(2.70±0.35)％,P ＜0.05]；高表达肿瘤干细胞标志CD133、CD166、Lgr5和间质分子波形蛋白(Vimentin)及Snail而低表达上皮细胞标志ZO-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.
목적 무혈청현부배양인결장암DLD-1세포형성세포구,검측기종류간세포표지표체변화,병탐토구세포발생상피간질전화여종류간세포급종류악성행위지간적련계.방법 재첨가생장인자적무혈청배양기중배양DLD-1세포형성구세포,운용Transwell실험검측세포적천이、침습능력변화；라서피하성류화연경지극륭형성실험분별검측체내외성류능력변화；류식세포술검측세포중CD133표체변화；실시정량취합매련반응(Real-time PCR),Western blot검측CD133、CD166、부함량안산중복서렬G-단백우련수체5(Lgr5)급상피간질전화관건기인표체변화.결과 구세포중CD133+세포명현증가[(0.90±0.03)％비(2.70±0.35)％,P ＜0.05]；고표체종류간세포표지CD133、CD166、Lgr5화간질분자파형단백(Vimentin)급Snail이저표체상피세포표지ZO-1(P＜0.05).구세포적전이、침습、체내외성류능력명현증가(P＜0.05).결론 수연점개단백(E-cadherin)표체명현승고,단시결합체내외실험결과,아문인위:E-cadherin가능불시개도세포악성행위적독립인소,현부배양형성적결장암구세포이발생상피간질전화,표현출종류간세포적특정.
Objective To investigate the similarities between cancer stem cells and epithelial mesenchymal 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,leucine-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 respectively.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 migration,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,Ecadherin-the key mediator of EMT was higher expressed too.Conclusion Although the E-cadherin was increased expression,considered the experiments in vitro and in vivo,we thought that E-cadherin may be not the sole canse of the malignant files in spheroid cells.Moreover,the spheroid cells exhibit cancer stem cells properties and EMT Characteristics.