胃癌干细胞的分离鉴定以及干细胞特性研究

Isolation,Identification and Stemness Characteristics of Gastric Cancer Stem Cells

背景:肿瘤干细胞(CSCs)已成为当前肿瘤研究的热点之一,开展相关研究的首要问题是CSCs的分离和鉴定,悬浮培养法是分离CSCs的重要方法。目的:分离、鉴定胃癌干细胞(GCSCs)并评价其干细胞特性。方法:人胃癌细胞株MKN45、MGC803、SGC7901以含表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)的无血清培养基悬浮培养。对培养得到的第三代肿瘤球,以集落形成实验检测其克隆形成能力,免疫荧光法检测GCSCs标记物,细胞划痕实验和Transwell小室细胞侵袭实验检测其迁移、侵袭能力,CCK-8实验检测其对顺铂的耐药性。结果:培养得到的肿瘤球高表达干细胞标记物CD44、CD54,低表达胃壁细胞标记物H+/K+-ATPase,克隆形成能力、划痕愈合速度和Transwell小室穿膜细胞数明显大于或多于普通胃癌细胞,差异有统计学意义(P<0.05)。顺铂对肿瘤球的50%抑制浓度明显高于普通胃癌细胞。结论:应用无血清培养基悬浮培养法能成功获得GCSCs富集的肿瘤球;肿瘤球具有较强的自我更新、增殖、迁移、侵袭能力和多向分化潜能,对化疗药物更易产生耐药性。

Background：Recently,cancer stem cells（ CSCs）has become a hot topic in cancer research. For this research topic,the primary problem is the isolation and identification of CSCs,and suspension culture is considered to be an important method for CSCs isolation. Aims：To study the isolation and identification of gastric cancer stem cells（ GCSCs） and evaluate its stemness characteristics. Methods：GCSCs-like cells were enriched through spheroid formation by suspending the human gastric cancer cell lines MKN45,MGC803 and SGC7901 in serum-free medium containing epidermal growth factor（ EGF）and basic fibroblast growth factor（ bFGF）. The third generation tumor spheres obtained were tested for the ability of colony forming;its stemness markers were identified by immunofluorescent staining,and migration and invasion capabilities were assessed by wound scratch test and Transwell assay,respectively. Growth inhibition of tumor spheres in response to cisplatin was assessed by CCK-8 assay. Results：High expressions of stem cell markers CD44 and CD54 were observed in the tumor spheres,but gastric parietal cell marker H＋ /K＋-ATPase was lowly expressed. Compared with parental gastric cancer cells,tumor spheres had stronger colony forming ability（P〈0. 05）;Furthermore,healing of wound scratch was accelerated（P〈0. 05）,and number of invasive cells in Transwell chamber was increased in tumor spheres（P〈0. 05）. The 50% inhibition concentration of cisplatin to tumor spheres increased significantly when compared with parental gastric cancer cells. Conclusions：GCSCs enriched tumor spheres can be obtained by suspension culture with serum-free medium. The tumor spheres possess enhanced capabilities of self-renewal,proliferation,migration,invasion and potential of multilineage differentiation;meanwhile,it is more resistant to chemotherapy.