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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 culture ovarian cancer stem cells from human ovarian cancer cell line A2780 and identify its characteristics. Methods: A2780 cells were digested and centrifuged to make a single cell suspension. The cells were suspended in SFM containing growth factors to obtain tumor cell microspheres. Flow cytometry was used to detect the expression of CD44 and CD133 on cell surface. Transwell chamber invasion assay was used to detect the invasion ability of tumor microspheres in vitro. CCK-8 was used to detect the proliferation ability of microspheres and the doubling time of the two cells was compared. The tumor microspheres were induced to differentiate in serum-containing medium and observed Its morphological changes. Results: A2780 could form tumor cell microspheres which can grow in suspension and stably pass in SFM. Compared with adherent tumor cells, microspheres highly expressed stem cell markers CD44 and CD133 (P <0.05), and had stronger invasiveness in vitro (t = 9.354, P <0.05) and proliferative ability (t = 0.05), and shorter doubling time (t = 3.773, P <0.05), which could differentiate into normal tumor cells in serum environment. CONCLUSIONS: Ovarian cancer tumor cell microspheres are obtained from the SFM-containing ovarian cancer cell line A2780 containing growth factors. The tumor cells are enriched with tumor stem cells.