人鼻咽癌放射抗拒性肿瘤干细胞的分离、筛选与鉴定
Screening and identification of tumor stem cells in radioresistance human nasopharyngeal carcinoma

摘要：目的 分离、筛选、鉴定人鼻咽癌CNE-2及放射耐受CNE-2R的肿瘤干细胞。方法 免疫磁珠技术分选CNE-2及CNE-2R中鼻咽癌CD133+干细胞，分别用无血清培养法、平板克隆法、裸鼠成瘤试验检测分选的CD133+细胞体外增殖能力、克隆形成能力及成瘤能力，HE染色和电镜超微结构观察形态学变化。结果 CNE-2及CNE-2R细胞中CD133+表达分别为(1.79±0.16)%和(2.63±0.72)%；CNE-2-CD133+细胞及CNE-2R-CD133+细胞形成肿瘤干细胞球及体外增殖能力远高于CD133-表达者；CNE-2R-CD133+细胞克隆形成能力较CNE-2-CD133+细胞明显升高，而且CNE-2R-CD133+细胞的克隆形态与CNE-2-CD133+细胞相比较明显增大；形态学发现CNE-2R-CD133+细胞核大，异型，核仁大，细胞器肥大。结论 分离、筛选出的鼻咽CNE-2R-CD133+肿瘤干细胞能形成干细胞球，其增殖能力、裸鼠成瘤能力更强，能为进一步探讨鼻咽癌放射抗拒的机制提供研究基础。
ABSTRACT: Objective To isolate, screen and identify human nasopharyngeal carcinoma CNE-2 and radioresistance CNE-2R cancer stem cells. Methods The CNE-2 and CNE-2R CD133+ stem cells were sorted with flow cytometry and immunomagnetic beads. The CNE-2-CD133+ and CNE-2R-CD133+ stem cells were detected for the abilities of tumor sphere formation and proliferation in vitro. The clonogenic ability of CD133+ stem cell was detected by plate clone formation assay and tumorigenicity was detected by nude mice test in vivo. Morphological changes were observed with HE staining and electron microscopy. Results CD133+ was expressed (1.79±0.16)% and (2.63±0.72)% in CNE-2 and CNE-2R cells, respectively. The clonogenic ability and tumor sphere formation ability of CNE-2R CD133+ cells were much greater than those of CD133- cells. The ability of proliferation of CD133+ cell sorted in vitro, especially CNE-2R-CD133+ cells, was stronger than that of CD133- cells. The morphological results showed that the nucleus of CNE-2R-CD133+ cells was larger and irregular with big nucleolus and organelles. Conclusion Nasopharyngeal CNE-2R-CD133+ tumor stem cells isolated and screened can form tumor sphere. The abilities of proliferation and tumorigenesis are stronger. The identification of CNE-2R TSC provides foundation for further studies on the mechanism of radioresistance of nasopharyngeal carcinoma