摘要
目的目前分离和鉴定鼻咽癌干细胞的方法仍不成熟。探索鼻咽癌细胞系干细胞微球体培养方法,并对CNE-2细胞微球体是否具有肿瘤干细胞生物特性(干性)进行鉴定。方法人鼻咽癌细胞CNE2、C666-1细胞在含生长因子的无血清培养基(serum-free medium,SFM)中悬浮培养。流式细胞术、Transwell小室实验、裸鼠成瘤实验、分别检测CNE2贴壁细胞(CNE2 monolayer,CNE2-MN)及CNE2微球体细胞(CNE2 sphere cells,CNE2-SC)CD133+标记细胞比例,体外侵袭能力、体内成瘤能力;CNE2-SC在含血清培养基中贴壁培养观察其分化能力;实时荧光定量PCR分析CNE2-MN及CNE2-SC相关干性基因Bmi-1、Oct4、Twist1RNA的表达。结果 2种细胞系均能在特殊配制的SFM中形成可以稳定传代的微球体,SFM新鲜配制、用细胞分离剂Accutase替代胰酶传代以及保持细胞的悬浮状态均有利于微球体的形成和增殖;在含血清培养基中培养能贴壁分化成CNE2-MN而无明显差异;CNE2-SC中CD133+细胞比例(98.79%)显著高于CNE2-MN(0.98%),差异有统计学意义(P<0.01);与CNE2-MN相比,CNE2-SC高表达干细胞相关基因Bmi-1、Oct4、Twist1及EMT标志物N-cadherin、Vimentin。Transwell小室实验示CNE2-SC与CNE2-MN的穿膜细胞数分别为(122±6)个/视野及(36±7)个/视野(P<0.05);裸鼠成瘤实验示1×104个CNE2-SC 3周内即能成瘤,成瘤率33.3%,而CNE2-MN不能成瘤,1×105个CNE2-SC与CNE2-MN成瘤体积比为(1.750±0.613)cm3vs(0.457±0.291)cm3(P<0.05),而1×106个以上2种细胞成瘤体积比为(2.332±0.549)cm3vs(0.669±0.278)cm3(P<0.01)。结论利用特制SFM悬浮培养法可得到鼻咽癌CNE2-SC,这种微球体富集了肿瘤干细胞,将为后续鼻咽癌干细胞研究打下基础。
Objective At present, the methods of separating and identifying nasopharyngeal cancer stem cells are not yet mature.This study was to explore the methods of culturing nasopharyngeal cancer stem cell microspheres and identify the cancerous stem cell biological features of CNE-2 cell microspheres. Methods We conducted suspension culture of human nasopharyngeal cancer CNE2 cells and C666-1 cells in serum-free medium （ SFM） containing growth factors.Then we measured the proportion of CD133 ＋cells in CNE2 monolayer （ CNE2-MN） and CNE2 microsphere cells （ CNE2-SC） by flow cytometry, determined their in vitro invasiveness through Transwell chamber experiments, and detected their in vivo tumorigenicity via nude mouse experiments.We ob-served the differentiation potency of the CNE2-SCs in the adherent-cultured serum-containing medium and detected the expressions of the cancer stem cell-related genes Bmi-1, Oct4, and Twist1 in CNE2-MN and CNE2-SCs by flow cytometry and RT-PCR analysis. Results In the special blend of SFM, both of the cell lines can form microspheres that can be stably transferred.Fresh SFM prepara-tion, substituting cell separation agent Accutase for pancreatic enzyme for transfer, and maintaining the state of cell suspension contrib-uted to the formation and proliferation of microspheres.Adherent culture with serum-containing medium induced the differentiation of CNE2-MN cells, which exhibited no significant difference from the CNE2 microsphere cells.The CD133 ＋cells accounted for 98.79%in the CNE2 microspheres, significantly higher than 0.98%in the CNE2 cells （P〈0.01）.Compared with the CNE2-MN cells, the CNE2-SCs showed highly increased expressions of Bmi-1, Oct4, and Twist1 （P〈0.01）.The numbers of membrane-penetrating cells in the CNE2-SCs and CNE2-MN cells were 122 ±6 and 36 ±7 per visual field, the former with a stronger invasive ability than the latter genesis of 1 ×106 CNE2-SCs vs that of the same number of CNE2 -MNcells was （2.332 ±0.549） cm 3 sv （0.669 ±0 .27
出处
《医学研究生学报》
CAS
北大核心
2014年第11期1133-1138,共6页
Journal of Medical Postgraduates
基金
国家自然科学基金(81171365)
关键词
鼻咽癌
肿瘤干细胞
微球体
悬浮培养
无血清培养基
Nasopharyngeal cancer
Cancer stem cell
Microsphere
Suspension culture
Serum-free medium
