人胆囊癌SGC996细胞株侧群细胞的分离与体外培养

目的 分选人胆囊癌细胞株SGC996的侧群细胞,并行体外培养,初步验证其干细胞的特性.方法 应用流式细胞仪分选人胆囊癌细胞株SGC996的侧群细胞和主群细胞,将两群细胞分别在有血清和添加干细胞因子的无血清培养液条件下培养.应用噻唑蓝(MTT)法检测两群细胞在血清培养条件下的增殖能力.结果 人胆囊癌细胞株SGC996的侧群细胞分选比例仅为1.0％,在维拉帕米阻断后降至0.1％.侧群细胞在无血清培养液中能形成干细胞样“肿瘤球”,但主群细胞在该条件下生长能力较差,甚至死亡.MTT结果显示,在有血清培养条件下,侧群细胞在第5、6、7天的吸光度值分别为1.26±0.15、1.73±0.18、2.21±0.17,较主群细胞的0.95±0.06、1.31±0.10、1.86±0.11,各组差异均有统计学意义(t=3.30、3.54、3.03,P＜0.05).结论 人胆囊癌细胞株SGC996中存在侧群细胞,这群细胞在体外培养能形成干细胞样“肿瘤球”,表现出更强的体外增殖能力,为胆囊癌干细胞进一步研究提供理论基础.

Isolation of side population cells in human carcinoma of gallbladder SGC996 cells and culture in vitro

Objective To isolate side population cells and non-side population cells from human carcinoma of gallbladder SGC996 cell line,and cultured the two subpopulations in vitro,and prehminarily verified its characteristics of stem cells.Methods Flow cytometry was used to isolate side population cells and main population cells and cultured the two subpopulations in serum-free culture solution with epidermal growth factor (EGF),basic fibroblast growth factor (bFGF) and LIF and in serum culture solution respectively.Multiplication capacity in vitro of the two subpopulations was observed in serum culture solution by methyl thiazol tetrazolium (MTT).Results The proportion of side population cells was 1.0％ by flow cytometry,by addition of verapamil,it was 0.1％,which was greatly decreased.Side population cells exhibited higher multiplication capacity and were able to form “tumor spheres” in serum-free culture solution;while main population cells showed lower multiplication capacity and even died in the same condition.The result by MTT showed that,in serum culture solution the spectrophotometry values of side population cells on the 5th day,6th day and 7th day were 1.26 ±0.15,1.73 ±0.18 and 2.21 ±0.17,while the main population cells 0.95 ±0.06,1.31 ±0.10 and 1.86 ±0.11.Each group was significantly different (t =3.30,3.54,3.03,P ＜ 0.05 ).Conclusion Human carcinoma of gallbladder SGC996 cell line contained a small subpopulation of cells that could form “tumor spheres” and exhibited higher multiplication capacity in vitro,which provided further theoretical foundation for gallbladder cancer stem cells.