人鼻咽癌干细胞微球体培养并鉴定其生物特性

发布时间：2018-04-04 22:16

  本文选题：鼻咽癌　切入点：肿瘤干细胞　出处：《医学研究生学报》2014年11期

【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(P0.01);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(P0.05);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(P0.05),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(P0.01)。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。
[Abstract]:Objective the method of isolation and identification of nasopharyngeal carcinoma stem cells is still immature.To explore the method of cell microsphere culture of nasopharyngeal carcinoma (NPC) cell line and to identify whether the microsphere of CNE-2 cell has the biological characteristics of tumor stem cells (dryness).Methods Human nasopharyngeal carcinoma cell line CNE2C666-1 was cultured in serum-free medium containing growth factor (SFM).CNE2 adherent cells CNE2 monolayerus CNE2-MNs and CNE2 microspheres somatic cells CNE2 sphere cells CNE2-SCE2-CD133 were detected by flow cytometry and tumorigenesis in nude mice, respectively, and the invasiveness of CNE2 cells in vitro was detected.The differentiation ability of CNE2-SC was observed by adherent culture in serum-containing medium in vivo, and the expression of CNE2-MN and CNE2-SC related dry gene Bmi-1Oct4 Twist1 mRNA was analyzed by real-time fluorescence quantitative PCR.Results two kinds of cell lines could form stable microspheres in specially prepared SFM. It was beneficial for the formation and proliferation of microspheres to replace trypsin passage with Accutase and to maintain the suspension state of the cells.鍦ㄥ惈琛，

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