喉癌CD133+侧群细胞的分离及体外生物学特性分析

目的 探索喉癌肿瘤干细胞的分选方法。方法 联合应用藻红蛋白标记的CD133单抗、侧群细胞（side population,SP）分选及流式细胞仪荧光活化细胞分选技术(fluorescence activated cell sorting,FACS)检测及分选喉癌Hep-2细胞系中的CD133+ SP及CD133 - SP细胞,在体外对2个亚群细胞进行肿瘤干细胞特性相关的增殖、分化、球体形成及药物敏感性试验并进行比较。结果 CD133+ SP细胞在喉癌Hep-2细胞系中占(0.30±0.12)％,远低于CD133+亚群(3.15±0.83)％及SP亚群(17.1±2.0)％。在体外增殖实验中CD133+SP较CD133- SP细胞表现出更强的增殖能力;纯化的CD133+ SP细胞体外培养后可以分化出CD133-SP细胞及非SP细胞,而CD133 - SP却不能分化成CD133+SP;CD133+SP在含生长因子的无血清培养基中可以形成悬浮生长的球体;CD133+ SP较CD133-SP有更强的化疗抵抗力。结论 CD133+SP较CD133-SP具备更明显的肿瘤干细胞特性,较CD133+亚群及SP亚群更有效地富集了喉癌肿瘤干细胞,可作为喉癌肿瘤干细胞研究的理想候选亚群。

Isolation and in vitro characterization of CD133(+) side population cells from laryngeal cancer cell line

Objective To investigate an approach enriching cancer stem cells (CSCs) more effectively from laryngeal cancer cell line. Methods CD133 + SP and CD133- SP subpopulation was detected and isolated from Hep-2 cell line using Hoechst33342 dye and phycoerythrin ( PE ) -conjugated CD133 monoclonal antibody assisted by fluorescence activated cell sorting technology. Sorted CD133 + SP and CD133- SP cells were compared in CSCs-related assays including proliferation, differentiation, spheroid formation and drug sensitivity. Results CD133 + SP cells accounted for a very small fraction of(0.30 ±0. 12)％ in Hep-2 cell line, far less than the proportion of CD133 + subgroup and side population subgroup,which were (3.15 ±0.83)％ and (17.1 ±2.0)％ respectively. Intriguingly, CD133+SP cells proliferated much faster than CD133 - SP cells in RPMI1640 and gave rise to CD133 - SP cells and other heterogeneous cells that formed the bulk of the tumor. In contrast, CD133- SP cells were not able to differentiate into CD133 + SP cells. In serum-free medium CD133 + SP cells grew as spherical clusters and remained floating.In addition, CD133 + SP cells manifested the marked resistance to chemotherapy than CD133- SP cells.Conclusions Compared with CD133- SP cells, CD133+ SP subpopulation exhibited extraordinary cancer stem-like properties, were enriched for cancer stem cells more effectively and might serve as an ideal putative candidate for CSCs research in laryngeal cancer.