辐射与悬浮球培养联合纯化乳腺癌干细胞

Suspension sphere culture combined with irradiation for purifying breast cancer stem cells

目的:利用悬浮球培养联合不同辐射模式,高度纯化人乳腺癌MCF-7细胞中辐射抵抗的CD44+/CD24-/low乳腺癌干细胞。方法:利用含细胞因子的无血清培养体系培养MCF-7细胞,将培养形成的球囊细胞连续传代,选择第2代球囊细胞分别行2Gy×4次及单次8Gy照射。利用滤网收集存活细胞生成的微球囊,流式细胞仪检测纯化后微球囊中CD44+/CD24-/low细胞的含量,克隆形成检测及单击多靶模型曲线拟合分析纯化后微球囊细胞的辐射敏感性。结果:单纯悬浮球培养与联合X线照射组之间CD44+/CD24-/low细胞的含量差异存在显著性(P﹤0.05),联合组高度纯化CD44+/CD24-/low细胞;多次分割及单次大剂量照射对CD44+/CD24-/low细胞的富集作用差异无显著性(P=0.303)。纯化细胞照射后存活分数显著高于贴壁MCF-7细胞。结论:辐射联合悬浮球培养进一步纯化辐射抵抗的CD44+/CD24-/low乳腺癌干细胞的方法是可行的。

Objective To highly purifying CD44＋/ CD24-/low breast cancer stem cells resistant to irradiation in cell line MCF-7 by using suspension sphere culture combined with different patterns of irradiation.Methods Human breast cancer MCF-7 cells were cultured in serum-free media containing cytokines.The second generation of saccular cells were irradiated by 4 fraction of 2 Gy and a single dose of 8 Gy, respectively.The tiny saccule produced by the surviving cells exposed to irradiation were collected.The expressions of CD44 and CD24 were measured by flow cytometry.Clone formation assay was used to assess radiosensitivity of the purified saccular cells.Results The content of CD44＋/ CD24-/low cells differed significantly between suspension sphere culture alone and a combination of the culture and irradiation（P 0.05）.The difference in the increased proportion induced by 4 fraction of 2 Gy or a single dose of 8Gy was not significant（P = 0.303）.The purified cells had a higher survival fraction after irradiation than its parent MCF-7 cells.Conclusions It is feasible to combine irradiation with suspension sphere culture for further purifying CD44＋/ CD24-/ low breast cancer stem cells resistant to irradiation.