人卵巢癌细胞系A2780肿瘤干细胞分离培养与鉴定

Isolation and identification of tumor stem cells from human ovarian carcinoma cell line A2780

目的:从人卵巢癌细胞系A2780中分离培养卵巢癌干细胞,并对其特性进行鉴定。方法:将A2780细胞消化,离心制成单细胞悬液,悬浮于含有生长因子的无血清培养基(SFM)中获得肿瘤细胞微球体,流式细胞仪检测细胞表面分子标志CD44和CD133的表达;Transwell小室侵袭实验检测肿瘤微球体的体外侵袭能力;CCK-8检测微球体细胞的增殖能力,比较两种细胞的倍增时间;将肿瘤微球体置于含有血清的培养基使其诱导分化,并观察其形态学变化。结果:A2780可在SFM中形成可悬浮生长、稳定传代的肿瘤细胞微球体。与贴壁的肿瘤细胞相比,微球体高表达干细胞标志CD44和CD133(P<0.05),具有更强的体外侵袭力(t=9.354,P<0.05)和增殖能力(t=13.682,P<0.05),及更短的倍增时间(t=3.773,P<0.05),在血清环境下可分化为普通的肿瘤细胞。结论:用含有生长因子的SFM悬浮卵巢癌细胞系A2780可获得卵巢癌肿瘤细胞微球体,此细胞球体中富集有肿瘤干细胞。

OBJECTIVE： To isolate and cultivate ovarian tumor stem ceils from human ovarian tumor cell line A2780, and identify their biological properties. METHODS： Cells A2780 were dissociated into single tumor cells, suspended in specific serum-free medium （SFM） to acquire ovarian tumor stem cell spheres. The expression of cell surface markers CD44 and CD133 was detected with flow-cytometry. Transwell inva- sive method was utilized to analyze the invasive ability in vitro and CCK-8 assays were performed to detect their proliferating potentiality. To compare their doubling time, and observe the inducing differentiation and cytologic and morphologie characteristics of the stem cell spheres. RESULTS： Nonadherent and serial-propagating tumor stem cells could be obtained from cell line A2780, which highly expressed stem cell markers CD44 and CD133 （P〈0. 05） ,with stronger invasive ability in vitro （t= 9. 354, P〈0.05） ,stronger propagating capability （t=13. 682, P〈0.05）and shorter doubling time （t=3. 773,P〈0.05）. They could differentiate normal tumor cells in serum supplemented medium condition. CONCLUSION： Ovarian tumor stem cell spheres can be obtained by serum-free suspended culture, which is enriched with this kind of stem cells.