人胃癌细胞系SGC-7901干细胞样细胞的富集与鉴定

Enrichment and Identification of Cancer Stem-like Cells from Human Gastric Cancer Line SGC-7901

【目的】富集/分离人胃癌细胞系SGC-7901干细胞样细胞,并评价其干细胞生物学特性。【方法】分别采用成球条件培养法和流式侧群(SP)细胞分选法分离SGC-7901干细胞样细胞,通过克隆形成实验、体内成瘤实验、化疗耐药实验鉴定SGC-7901细胞球、SP细胞的干细胞特性。【结果】(1)成球条件培养法:在含有表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)、B27、N-2的无血清条件培养基及超低吸附培养板的培养条件下,SGC-7901细胞能够成球悬浮生长,且可连续传代;克隆形成实验结果显示:SGC-7901细胞球克隆数高于SGC-7901细胞;体内成瘤实验显示:裸鼠移植相同数量级的SGC-7901细胞球和SGC-7901细胞,SGC-7901细胞球成瘤率高于SGC-7901细胞;化疗耐药实验结果显示:在相同浓度下,3种化疗药(5-氟尿嘧啶、盐酸多柔比星、顺铂)对SGC-7901细胞球的平均抑制率均低于SGC-7901细胞。(2)侧群分选法:Hoechst33342浓度为2.5μg/mL,SGC-7901细胞SP细胞分选率为4.74%,维拉帕米对照组SP细胞分选率为0.19%,差异显著(P <0.01);克隆形成实验与体内成瘤实验显示:SP与非侧群(non-SP)细胞克隆形成能力与致瘤性无显著性差异(P>0.05)。【结论】在超低吸附培养板和无血清条件培养基培养的SGC-7901细胞球具有肿瘤干细胞特性;SGC-7901细胞存在着SP亚群,但其肿瘤干细胞特性不明显。

Objective To enrich and isolate the cancer stem-like cells from human gastric cancer cell line SGC-7901,and to evaluate their biological characteristics.Methods Gastric cancer stem-like cells SGC-7901 were isolated by the method of sphere-forming cultivation and the method of side population(SP)cells selected by flow cytometry,respectively.Colony formation assay,in vivo tumorigenicity assay and multidrug resistance assay were used to identify the stem cell properties of SGC-7901 cell spheres and SP cells.Results(1)Sphere-forming cultivation method:SGC-7901 grew with sphere-forming and suspending features in super-low adherent culture plate under the condition of serum-free culture medium containing epidermal growth factor(EGF),basic fibroblast growth factor(bFGF),B27 and N-2,and can be subcultivated continuously.The results of colony formation assay showed that the number of colonyies of SGC-7901 cell spheres was higher than that of SGC-7901cells.The results of in vivo tumorigenicity assay showed that when the nude rats were transplanted with the same number of SGC-7901 cell spheres or SGC-7901 cells,the rate of tumor formation in SGC-7901 cell spheres was higher than that in SGC-7901 cells.The results of multidrug resistance study showed that the average inhibition rate of chemotherapeutic drugs(including 5-fluorouracil,doxorubicin hydrochloride and cisplatin)at the same concentration for SGC-7901 cell spheres was lower than that for SGC-7901 cells.(2)SP cell selection results showed that the concentration of Hoechst33342 was chosen at 2.5μg/mL.At 2.5μg/mL of Hoechst33342,SP cell selection rate for SGC-7901 cells was 4.74%,and the SP cell selection rate was 0.19%in velapami control group,the difference being significant(P<0.01).The results of colony formation assay and in vivo tumor formation assay showed that there was no difference in activity of colony formation and carcinogenesis between SP and non-SP SGC-7901 cells(P>0.05).Conclusion SGC-7901 cell spheres cultivated in super-low adherent culture plate under the condition of serum-free culture medium have the characteristics of cancer stem-like cells.There exist SP cells in SGC-7901 cells,but their characteristics of cancer stem cells are not apparent.