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目的:从人卵巢癌细胞株SKOV-3中分离培养卵巢癌干细胞,并鉴定其生物学性质。方法:卵巢癌SKOV-3细胞在含有生长因子的无血清培养基(SFM)中悬浮培养得到肿瘤细胞球。流式细胞术、Transwell小室侵袭实验分别检测SKOV-3细胞及其细胞球CD44和CD117的表达,筛选细胞表面标记物和观察体外侵袭能力;将肿瘤细胞球传代扩增,并用含血清培养基(SSM)培养促使其分化,并将细胞球和普通SKOV-3细胞分别接种于96孔板,CCK-8法检测增殖能力及其耐药性。结果:卵巢癌SKOV-3细胞能在SFM中形成可以稳定传代的细胞球,并显示很强的自我更新和增殖能力,含血清环境能够诱导其分化而贴壁生长;肿瘤细胞球中高表达CD44和CD117抗原标记,特别是CD44,其侵袭能力显著高于正常SKOV-3细胞(P<0.05);细胞球对顺铂具有更强的耐药性,将顺铂作用于细胞球及贴壁分化细胞24,48及72h后,细胞球抑制率分别为2.59%,8.43%,12.08%,显著低于分化贴壁细胞(抑制率分别为10.73%,28.13%,39.17%),分别比较,差异均有统计学意义(P<0.05)。结论:采用无血清悬浮培养法从人卵巢癌细胞系中获取的细胞球,可能含有一小部分具有增殖和分化能力的卵巢癌干细胞,多表达CD44和CD117抗原标记,且CD44更具有特异性。
OBJECTIVE: To isolate and culture ovarian cancer stem cells from human ovarian cancer cell line SKOV-3 and identify their biological properties. Methods: Ovarian cancer SKOV-3 cells were cultured in suspension in growth medium containing serum-free medium (SFM) to obtain tumor cell spheres. Flow cytometry and Transwell chamber invasion assay were used to detect the expression of CD44 and CD117 in SKOV-3 cells and their cell spheres. The cell surface markers were screened and the invasion ability in vitro was observed. The spheres of tumor cells were subcultured and expanded with serum-containing medium SSM) to promote their differentiation, and the cell spheres and normal SKOV-3 cells were seeded in 96-well plates respectively. The proliferation and drug resistance of CCK-8 cells were detected by CCK-8 assay. Results: SKOV-3 cells in ovarian cancer could form stable cell spheres in SFM and showed strong ability of self-renewal and proliferation. Serum-containing environment induced differentiation and adherent growth of tumor cells. High expression of CD44 The CD117 antigen marker, especially CD44, had significantly higher invasiveness than that of normal SKOV-3 cells (P <0.05). The cytoplasm was more resistant to cisplatin, and cisplatin could act on the cell spheres and adherent differentiated cells After 24, 48 and 72 hours, the inhibitory rates were 2.59%, 8.43% and 12.08%, respectively, which were significantly lower than those of adherent cells (inhibition rates were 10.73%, 28.13% and 39.17%, respectively) Statistical significance (P <0.05). CONCLUSION: The spheres obtained from human ovarian cancer cell lines using serum-free suspension culture may contain a small number of ovarian cancer stem cells with proliferative and differentiation ability, more expression of CD44 and CD117 antigen markers, and more specific of CD44.