长程无血清培养人乳腺癌细胞球的生物学特性

目的:从人乳腺癌原代细胞中分离培养乳腺癌干/祖细胞,并通过体外连续传代培养,研究其体外增殖、分化等生物学特性.方法:应用非黏附性乳腺球(mammosphere,MS)悬浮培养法富集乳腺癌干/祖细胞后,通过连续的单克隆实验检测乳腺球细胞(mammosphere-derived cells,MSDC)的克隆形成和自我更新能力,并经血清诱导后观察其分化能力.FCM法检测CD44~+/CD24~(-/low)细胞的含量.结果: 接种于含生长因子无血清培养液的原发乳腺癌标本细胞均有MS生成,内含具有干细胞特征的肿瘤细胞.随着培养中传代次数的增加,贴壁分化的细胞增多,形成的MS减少,球体变小,且更易贴壁分化.CD44~+/CD24~(-/low)细胞仅存在于基底样乳腺癌标本.来自不同标本的MS的生物学特性表现出一定差异.结论:具有自我更新、无限增殖和多向分化等干细胞样特性的肿瘤细胞在人乳腺癌组织中广泛存在,其生物学行为可能因标本来源不同有所差异,也会因环境因素的作用而改变.

Biological characteristics of human breast cancer mammospheres cultured in serum-free medium over long term

Objective:To isolate breast cancer stem/progenitor cells from human breast cancer and study their proliferation and differentiation biological characteristics over long-term passages in vitro. Methods:Human breast cancer stem/progenitor cells were enriched in suspension cultures as nonadherent mammospheres(MS). Serial sphere formation assay was performed to determine self-renewal ability of mammosphere-derived cells (MSDC). Differentiation was induced by culturing MSDC in DMEM-F12 supplemented with serum but without growth factors. The ratio of CD44~+/CD24~(-/low) cell population was evaluated by flow cytometry(FCM). Results:The mammospheres were formed after inoculation of primary breast cancer cells into culcutre medium with growth factors but without serum. The mammospheres contained undifferentiated cells similar to stem cells, which had self-renewal and extensive proliferation capabilities. With increasing passages, the cells tended to adhere and differentiate. The number of adhering and differentiating cells increased, and the amount and size of mammospheres decreased. The CD44~+/CD24~(-/low) cell population was enriched in the basal-like molecular subtype of human breast tumors. The biological behaviors of mammospheres varied between different specimens.Conclusion:Cancer cells with stem cell properties of self-renewal, indefinite proliferation and multi-lineage differentiation widely existed in human breast cancer tissues. The biological behaviors varied because of different origin of specimens and changed under the effects of environmental factors.