人上皮性卵巢癌细胞系侧群细胞的肿瘤干细胞样细胞生物学特性及膜蛋白差异表达的鉴定

Identification of bio-functions of cancer stem-like cells and differentially expressed membrane proteins in side population cells from human epithelial ovarian cancer cell lines

目的鉴定上皮性卵巢癌细胞侧群细胞(SP细胞)肿瘤干细胞样细胞生物学特性,并检测侧群细胞内差异蛋白质的表达。方法流式分选上皮性卵巢癌细胞系SKOV3和A2780具有外泌Hochest33342染料生物学特性的侧群细胞,鉴定其肿瘤干细胞样细胞相关生物学特性。运用细胞培养稳定同位素标记(SILAC)的定量蛋白质组学技术,检测SP细胞中差异表达膜蛋白。结果运用流式分选技术,成功分选出上皮性卵巢癌细胞系SKOV3和A2780细胞占比0.5%的SP细胞,经过多次分选,SP细胞比例逐步升高,并能够逐步分化成非SP细胞,说明SP细胞具有一定的增殖与分化能力。体外实验证实SP细胞具有更强的细胞克隆形成能力,且对紫杉醇、顺铂等化疗药物敏感性差;反转录PCR实验显示SP细胞内干细胞相关基因(如Oct-4、β-catenin、ABCG2)表达水平升高。此外,SP细胞具有较强的小鼠体内成瘤能力。运用SILAC技术,以非SP(NSP)细胞作为对照,检测出SKVO3和A2780细胞SP细胞内差异表达膜蛋白分别1561和1989个,以SKOV3为基准,两种SP细胞共同差异表达上调蛋白12种,共同差异表达下调蛋白13种。结论上皮性卵巢癌细胞系SP细胞具有肿瘤干细胞样细胞生物学特性,并检测出SP细胞差异表达膜蛋白,为后续深入探索上皮性卵巢癌肿瘤干细胞样细胞表面标记蛋白提供基础。

Objective To validate the stemness bio-function of side population(SP)cells from epithelial ovarian cancer cell lines and to detect the differentially expressed cellular membrane proteins in SP cells.Methods SP cells of human ovarian cancer cell lines SKOV3 and A2780 which secreted bisbenzimide Hoechst33342 and located as side population were sorted by flow cytometry.Bio-functions of stemness of SP cells were validated.Through stable isotope labeling by amino acids in cell culture(SILAC),differentially expressed membrane proteins in SPcells were detected.Results SP cells of SKOV3 and A2780 were sorted successfully.The ratio of SP cells was about 0.5%and increased significantly through repeated sorting.SP cells differentiated into non-SP(NSP)cells.Stemness of SP cells was validated in vitro,including cell clone forming,resistance to cisplatin and paclitaxel in NOD/SCID mice.SILAC technique was used to detect differentially expressed membrane proteins in SP cells.Results showed that 1561 and 1989 differentially expressed proteins were detected respectively in SKOV3 and A2780 SP cells.Among those significantly up-regulated proteins in SKOV3 SP cells,12 proteins were also up-regulated in A2780 SP cells.On the contrary,13 proteins were down-regulated both in SKOV3 and A2780 SP cells.Conclusions SP cells of human epithelial ovarian cancer cells show stemness.And differently expressed membrane proteins in SP cells were detected which would be the base of further screening surface biomarker of ovarian cancer stem-like cells.