CAJ | 학술논문

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）cm3 vs （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）cm3 vs （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。
목적：목전분리화감정비인암간세포적방법잉불성숙。탐색비인암세포계간세포미구체배양방법，병대CNE-2세포미구체시부구유종류간세포생물특성（간성）진행감정。방법인비인암세포CNE2、C666-1세포재함생장인자적무혈청배양기（ serum-free medium， SFM）중현부배양。류식세포술、Transwell소실실험、라서성류실험、분별검측CNE2첩벽세포（ CNE2 monolayer， CNE2-MN）급CNE2미구체세포（ CNE2 sphere cells， CNE2-SC） CD133＋표기세포비례，체외침습능력、체내성류능력；CNE2-SC재함혈청배양기중첩벽배양관찰기분화능력；실시형광정량PCR분석CNE2-MN급CNE2-SC상관간성기인Bmi-1、Oct4、Twist1RNA적표체。결과2충세포계균능재특수배제적SFM중형성가이은정전대적미구체， SFM신선배제、용세포분리제Accutase체대이매전대이급보지세포적현부상태균유리우미구체적형성화증식；재함혈청배양기중배양능첩벽분화성CNE2-MN이무명현차이；CNE2-SC중CD133＋세포비례（98．79％）현저고우CNE2-MN（0．98％），차이유통계학의의（ P＜0．01）；여CNE2-MN상비，CNE2-SC고표체간세포상관기인Bmi-1、Oct4、Twist1급EMT표지물N-cadherin、Vimentin。 Transwell소실실험시CNE2-SC여CNE2-MN적천막세포수분별위（122±6）개／시야급（36±7）개／시야（P＜0．05）；라서성류실험시1×104개CNE2-SC 3주내즉능성류，성류솔33．3％，이CNE2-MN불능성류，1×105개CNE2-SC여CNE2-MN성류체적비위（1．750±0．613）cm3 vs （0．457±0．291）cm3（P＜0．05），이1×106개이상2충세포성류체적비위（2．332±0．549）cm3 vs （0．669±0．278）cm3（P＜0．01）。결론이용특제SFM현부배양법가득도비인암CNE2-SC，저충미구체부집료종류간세포，장위후속비인암간세포연구타하기출。
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 .278) cm3 ( P<0.01). Conclusion Using suspension culture with a specially prepared SFM, nasopharyngeal cancer CNE2 microsphere cells can be obtained, in which there are large numbers of cancer stem cells.This culture method may provide a base for further studies of naso-pharyngeal cancer stem cells.