结直肠癌细胞系肿瘤干细胞相关亚群初步研究

Preliminary study on cancer stem-like cells(side population)in human colorectal cancer cell lines

目的:检测不同结直肠癌细胞系中存在肿瘤干细胞样亚群,探讨初步富集肿瘤干细胞相关亚群的方法。方法:采用流式细胞术检测结直肠癌细胞系LOVO、SW480、HCT116中的侧群细胞(Side population,SP)细胞,添加生长因子的无血清培养基(Serum-free medium,SFM)培养SW480初步分离,富集肿瘤干细胞样亚群,有限稀释实验,诱导分化,传代更新,含血清/无血清培养基交替培养证实肿瘤干细胞相关亚群的存在。结果:LOVO、SW480、HCT116细胞系中含SP细胞分别为1.0%,1.2%和1.3%;SW480细胞中约含有0.54%￣0.62%的细胞能够在无血清培养基中能够存活、增殖,形成悬浮的肿瘤细胞球;在SFM中加入血清可促使肿瘤干细胞相关细胞分化;肿瘤干细胞相关细胞可以连续传代,并可交替培养于含10%小牛血清培养基(Serum-supplied medium,SSM)和SFM中。结论:结直肠癌细胞系LOVO、SW480、HCT116中含SP细胞,SW480中的肿瘤干细胞相关亚群可以无血清培养基富集。

Objective： To investigate the content of cancer stem-like cells in different human colorectal cancer cell lines and explore the methods for enriching cancer stem-like cells in SWd80. Methods： The percentage of side population cells（SP）in human colorectal cancer cell lines LOVO,SW480,HCTI16 were detected by flow cytometry. SW480 cell line was cultivated in serum-free medium（SFM ）supplemented with growth factors and cancer stern-like cells reforming into floatingspheres were isolated. The isolated cancer stem-like cells were identified by limiting-dilution assay,differentiation assay,self-renewal assay, alternative cultivation assay. Results： The percentage of SP cells was 1.0% in LOVO,1.2% in SW480 and 1.3% in HCTII6. In the absence of serum,a minority（0.54%-0.62%） of cancer stem-like cells in SW480 cells survived,proliferated and assembled into the suspended tumor cell spheres. SW480 cancer stem-like cells possessed proliferative,self-renewal and differentiation potential,which were responsible for the floating tumor clone. Serum addition into SFM resulted in the proliferation of cancer stem-like cells;after several generations and ahernated cultivation in SSM and SFM,cancer stem-like cells maintained their characters. Conclusion： LOVO,SW480 and HCTI16 cell lines contain a tiny minority of SP cells with stem cell properiies.The cancer stem-like cells in SW480 line can be maintained in SFM using a floating culture method.