喉癌CD133^＋侧群细胞的分离及体外生物学特性分析

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

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CD133SP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。

Objective To investigate an approach enriching cancer stem cells （CSCs） more effectively from laryngeal cancer ceil 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.