人结直肠癌细胞系的成球培养方法及转移侵袭能力研究

Study of Cultivation and Metastasis-related Characteristics of Tumor Spheres Generated from Human Colorectal Cancer Cell Lines in Serum-free Medium

目的探索人结直肠癌细胞系形成肿瘤细胞球的培养方法及球体内间质化标志物的表达检测。方法结直肠癌细胞系SW480、SW620、LOVO、HT29及HCT116在添加了不同细胞生长因子的无血清培养基(SFM)中悬浮培养,传代更新,诱导分化。免疫荧光技术和RT-PCR分别检测细胞球中间质化标志物N-cadherin及Vimentin的表达情况。结果 5种细胞系均能在特制的SFM中形成细胞球,并稳定的传代增殖。在用血清谤导分化后,又重新贴壁生长,且与亲代细胞保持一致的细胞形态。细胞球中间质化标志物N-cadherin、Vimentin表述均上调。结论结直肠癌细胞系在低黏附及添加了细胞生长因子R-Spondin 1、Noggin的无血清环境中更容易形成悬浮生长的肿瘤细胞球,细胞球细胞比亲代细胞更具有转移侵袭能力。

Objective To study on the best cultivation method for cell spheres from colorectal cancer cell lines and examine the mesenchymal markers expression in those cell spheres. Methods Colorectal cancer cell lines SW480, SW620, LOVO, HT29 and HCT116 ceils were seeded in the serum free medium （SFM） supplemented with cell growth factors, the cancer cell spheres were subculture and induced to differentiate by serum supplemented medium, the expression of mesenchynial markers were investigated by immunofluorescence and RT-PCR. Results Cancer cell spheres could be generated from five colorectal cancer cell lines in SFM. These spheres were stable subculture and self-renewal, and were induced to differentiate by serum supplemented medium, these differentiated cell were adherence growth and kept the same morphol- ogy with the parental cells. These cancer cell spheres were founded expressing mesenchymal markers such as N-cadherin and Vimentin was higher than monolayer cells. Conclusion Tumor spheres can be generated and matained from colorectal cancer cell lines in SFM, and The metastasis ability of cell sphere was higher than monolayer cells.