人鼻咽癌细胞株中肿瘤干细胞分离鉴定方法及肿瘤多药耐药机制  

Separation and identification of tumor stem cells in human nasopharyngeal carcinoma cell lines and its mechanism of multidrug resistance

作　　者：刘超群 LIU Chaoqun(Department of Oncology,the Third Xiangya Hospital of Central South University,Changsha 410013,China)

机构地区：[1]中南大学湘雅三医院肿瘤科,湖南长沙410013

出　　处：《中国医学物理学杂志》2018年第8期956-961,共6页Chinese Journal of Medical Physics

基　　金：中国博士后科学基金(015T80890;2014M552167)

摘　　要：目的:研究人鼻咽癌细胞株中肿瘤干细胞分离鉴定方法及对肿瘤多药耐药机制。方法:取鼻咽癌细胞株(SUNE),进行细胞的培养、传代及鉴定,采用免疫荧光细胞化学技术联合流式细胞仪检测SUNE中CD133^-及CD133^+细胞体外分化能力。利用免疫磁珠分选技术完成CD133^+肿瘤细胞纯化,测定CD133^+细胞体外增殖能力,将其与未分选及CD133^-细胞进行比较。取顺铂、紫杉醇化疗药物,采用CCK^-8法测定CD133^+细胞耐药性。取10只4周龄ICR小鼠,皮下注射人鼻咽癌细胞株和普通细胞株,分析耐药性。结果:分离培养的SUNE在含有血清的培养基中呈贴壁生长,并且生长活跃,培养2~3 d后倒置显微镜下显示细胞呈梭形、扁平状,光泽度良好,培养2周左右细胞融合瓶底80.00%;流式细胞仪检测结果显示:约0.35%细胞表面膜存在抗原CD133。免疫细胞化学显示:SUNE细胞贴壁爬行,部分细胞能与SUNE中的CD133^-相互结合,荧光显微镜下显示呈现橙红色,球状;向经免疫磁珠分选后的细胞中加入培养基,细胞呈单细胞,球形,悬浮生长。随着培养时间的不断延长,细胞开始出现团状生长,体积增加,细胞数量增多,培养24 h后细胞开始贴壁生长,培养7 d后呈串状生长;CD133^+肿瘤细胞、CD133^-肿瘤细胞及未分选细胞随着时间的延长细胞均出现增长,且CD133^+肿瘤细胞增殖能力显著高于CD133^-肿瘤细胞及未分选细胞(P<0.05)。10只小鼠均可见瘤体形成,种植人鼻咽癌细胞株组的瘤体体积显著大于普通细胞株组(P<0.05)。结论:鼻咽癌干细胞中CD133^+对化疗药物的耐药性强。Objective To study the method for separating and identifying tumor stem cells in human nasopharyngeal carcinoma cell lines, and investigate the mechanism of multidrug resistance. Methods The cell culture, subculture and identification of nasopharyngeal carcinoma cell lines（SUNE） were carried out, and the in vitro differentiation ability of CD133^- cells and CD133^＋ cells in SUNE was detected with immunocytochemistry combined with flow cytometry. CD133^＋ tumor cells were purified with immunomagnetic cell separation technology. The in vitro proliferation ability of CD133^＋ cells was determined, and then compared with that of unsorted cells and CD133^-cells. The drug resistance of CD133^＋ cells in the treatment of cisplatin and paclitaxel was determined by CCK^-8 method. ten ICR mice aged 4 weeks were injected subcutaneously with human nasopharyngeal carcinoma cell lines and normal cell lines, and the mechanism of multidrug resistance was analyzed. Results The SUNE isolated and cultured in serum^-containing medium were adherent growth, with active growth. After SUNE being cultured for 2-3 d, inverted microscope showed that the cells were spindle, flat, with good gloss. After SUNE being cultured for about 2 weeks, the cell fusion bottle was80.00%; and with the use of flow cytometry, antigen CD133 was detected on about 0.35% of cell surface membrane.Immunocytochemistry showed that SUNE cells were adherent growth, some of which were combined with CD133^-in SUNE,appearing orange red and globose under fluorescence microscope. The cells after immunomagnetic cell separation were added to the medium, and the cells were single cells, spherical, in suspended growth. With the prolonging of culture time, cells began to appear nodular growth, increase in volume and cell quantity. The cells were adherent growth after being cultured for 24 h, and began to grow in clusters after 7 d of culture. The number of unsorted cells, CD133^-tumor cells and CD133^＋tumor cells were increased with the extension of time, and the pr

关 键 词：鼻咽癌 恶性肿瘤 肿瘤干细胞 分离鉴定 多药耐药 免疫荧光细胞化学技术 流式细胞仪 鼻咽癌细胞株 体外分化 免疫磁珠分选技术 

分 类 号：R739.6[医药卫生—肿瘤]