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目的探讨腺样囊性癌细胞(adenoid cystic carcinoma cell,ACC)系中肿瘤干细胞的存在依据。方法采用单细胞体外培养法、免疫组化染色、免疫磁珠分离技术和动物实验等方法,分析常规培养的和经分离分群的不同细胞表型的 ACC-2的体外增殖、分化特点和裸鼠体内成瘤能力的差异。结果体外培养至第8天,ACC-2单细胞仅有4.5%分裂、增殖,并非全部分裂。CD44~+-CD24~-亚群占所有 ACC-2细胞的8.1%,其中仅有25.71%的细胞长期存活和持续分裂。CD44~-和 CD44~+-CD24~+细胞在体外培养条件下均无长期存活能力。不同表型的 ACC-2体外单细胞培养实验发现,CD44~+发生分裂的比例为8.4%,CD44~+-CD24~-肿瘤细胞发生分裂的比例为14.7%,CD44~-和CD44~+-CD24~+细胞未发生分裂。裸鼠成瘤实验显示,CD44~+-CD24~-的成瘤最低接种细胞数为1×10~3个/L,常规 ACC-2最低成瘤细胞数为1×10~5个/L,CD44~-和 CD44~+-CD24~+细胞则均无成瘤能力。免疫组化染色证实,CD44~+-CD24~-肿瘤细胞具有分化成其他表型肿瘤细胞的能力。结论细胞表面抗原为 CD44~+-CD24~-的肿瘤细胞仅占 ACC-2肿瘤细胞的极少部分,这些细胞具有极强的增殖能力和分化为其他表型肿瘤细胞的能力。ACC-2肿瘤细胞的增殖和成瘤能力源于 CD44~+-CD24~-ACC-2细胞亚群。ACC-2中存在肿瘤干细胞,而 CD44~+-CD24~-是 ACC-2肿瘤干细胞必须兼备的表面标志。
Objective To investigate the existence of tumor stem cells in adenoid cystic carcinoma cell (ACC) lines. Methods The single cell in vitro culture method, immunohistochemistry, immunomagnetic beads separation and animal experiments and other methods were used to analyze the proliferation and differentiation of ACC-2 cultured in vitro and isolated by different cell phenotypes in vitro. In vivo tumorigenic ability differences. Results In vitro cultured to day 8, ACC-2 single cell only 4.5% split, proliferation, not all split. The CD44 ~ + -CD24 ~ - subpopulation accounted for 8.1% of all ACC-2 cells, of which only 25.71% of the cells were long-lived and persistent. CD44 ~ - and CD44 ~ + -CD24 ~ + cells have no long-term viability in vitro. In vitro single cell culture of ACC-2 with different phenotypes showed that the percentage of CD44 ~ + cells dividing was 8.4%, the percentage of CD44 ~ + -CD24 ~ - cells dividing was 14.7%, CD44 ~ - and CD44 ~ + - CD24 ~ + cells did not divide. The tumorigenicity experiments of nude mice showed that the minimum number of tumorigenic cells inoculated with CD44 ~ + -CD24 ~ - was 1 × 10 ~ 3 / L, the number of the most mature ACC-2 cells was 1 × 10 ~ 5 / L, CD44 ~ - and CD44 ~ + -CD24 ~ + cells were no tumorigenic ability. Immunohistochemical staining confirmed that CD44 ~ + -CD24 ~ - tumor cells have the ability to differentiate into other phenotypic tumor cells. Conclusion Tumor cells with CD44 ~ + -CD24 ~ - cell surface antigen account for only a very small part of ACC-2 tumor cells. These cells have strong proliferative capacity and ability to differentiate into other phenotypic tumor cells. ACC-2 tumor cell proliferation and tumorigenic ability from CD44 ~ + -CD24 ~ -ACC-2 cell subsets. ACC-2 tumor stem cells exist, and CD44 ~ + -CD24 ~ - ACC-2 tumor stem cells must be a surface marker.