人卵巢癌干细胞的分离培养和初步鉴定  

Isolation and initial identification of human ovarian cancer stem cells.

作　　者：武加利[1] 纪新强[1] 刘佳[1] 张文卿[2] 

机构地区：[1]青岛大学医学院附属医院妇科,青岛266003 [2]青岛大学医学院病原生物教研室

出　　处：《现代妇产科进展》2010年第12期893-896,共4页Progress in Obstetrics and Gynecology

基　　金：山东省自然科学基金(No:Y2008c33)

摘　　要：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 identify ovarian cancer stem cells from human ovarian cancer cell line SKOV-3 and to identify their biological properties.Methods：SKOV-3 cells were cultured in serum free medium（SFM）with EGF and bFGF to derive tumor spheres.Flow cytometry analysis and cell invasion assay were applied to examine the cell surface marker（CD44,CD117） expression pattern and cell invasive ability of both tumor spheres and parental cells;Tumor spheres were then expanded and cultured in serum-containing medium to permit their differentiation.The proliferative capacity and drug resistance of tumor sphere-forming cells was tested by cell counting kit-8（CCK-8）assay.Results：Small number of floating tumor spheres were isolated and expanded in serum free medium（SFM）.These tumor spheres attached to the bottom of culture plates and began to differentiate in serum-supplemented medium（SSM）;CD44 and CD117 antigens were significantly higher in tumor spheres than those in the common SKOV-3 cells,and the invasive ability was increased than those of parental cells（P0.05）.By contrast with the differentiated cells under standard serum-containing culture conditions,these sphere-forming cells were more resistant to cisplatin after 24,48 and 72 hours of treatment（2.3% vs 9.1%,8.6% vs 22.8% and 18.7% vs 36.8%,P0.05）.Conclusion：The sphere-forming cells isolated from human ovarian cancer cell line have the characterization of cancer stem cells,and significantly express CD44、CD117 antigens.CD44 may be a specific antigen marker for the human ovarian cancer stem cells.

关 键 词：卵巢肿瘤 肿瘤干细胞 肿瘤球状体细胞 无血清培养基 悬浮培养 

分 类 号：R737.31[医药卫生—肿瘤]