鼻咽癌细胞株CNE-2Z中肿瘤干细胞样细胞的悬浮培养与鉴定  

Methodological exploration on enrichment and identification of cancer stem cell-like clles in nasopharyngeal carcinoma cell line CNE-2Z by serum-free suspension culture for

作　　者：肖调立[1,2] 田道法[1] 何迎春[1] 刘湘[1] 范婧莹[1] YANG PC 

机构地区：[1]湖南中医药大学中西医结合学院,长沙410007 [2]湖南省第二人民医院整形美容科 [3]McMaster university Canada

出　　处：《中国中西医结合耳鼻咽喉科杂志》2013年第1期16-21,共6页Chinese Journal of Otorhinolaryngology in Integrative Medicine

基　　金：国家自然科学基金(No.81072904;No.30973856;No.81273988);中国博士后基金(No.2011M501277);湖南省科技计划项目(No.2010SK3021);湖南省2011年研究生创新基金研究项目(No.CX2011B355);湖南省外国专家局引智项目(湘财社指2011[40]号);湖南省中医药科研项目(2010007;201142);湖南省中医五官科学重点学科;湖南省海外名师计划(湘教通2012-488)

摘　　要：目的探讨鼻咽癌细胞株CNE 2Z中肿瘤干细胞样细胞的富集与鉴定方法。方法鼻咽癌细胞株CNE 2Z细胞普通培养至对数生长期,不同密度细胞分别加入无血清培养液中继续培养,制备第9天无血清培养细胞爬片,CD133和CD44克隆抗体免疫组化染色,电镜观察超微结构。结果无血清悬浮培养至第9天时,以1×10~5/m1细胞密度组活细胞比率最高;普通培养细胞爬片的CD133和CD44细胞强阳性率分别是2.71%和3.00%,而无血清培养细胞爬片的CD133和CD44强阳性率分别为66.7%和67.00%(P<0.01),表现正相关(P<0.01);扫描电镜和透射电镜观察显示,阳性细胞表现特殊的超微结构特点。结论无血清悬浮培养法可富集鼻咽癌细胞中的肿瘤干细胞样细胞,CD133和CD44可作为其特异性表面标志物。Objective TTo investigate the methodology for enrichment and identification of cancer stem cell-like clles in nasopharyngeal carcinoma cell line CNE-2Z by serum-flee suspension culture for. Methods CNE-2Z cells were collected into logarithmic growth phase by routine culturing procedure at first. Then, they were transferred to serum-free culture medium in different density to prepare living cell slices on coverslips at the 9th day during the continuous culturing period. Followed were the procedures of immunohistochemical staining with CD 133 and CD44 moloclonal antibodies on these cell slices to determine the positive numbers on them. Electron microscopic observation was carried out on these cell samples to identify their ultrastructural features at last. Results Living cells ratio was highest at 9th day with the density of l^105/ml in these serum-free culturing system. Rates of positive cells to CD133 and CD44 were 2.71% and 3.00% on these cell slices in common culturing system, while they were 66.7% and 67.00% respectively on such coverslips in serum-free culturing system, with very significantly statistical significances （P〈0.01） and a positive correlation （P〈0.01） among them. It was found that these cancer stem-like cells held specific ultrastructural features as shown through scanning electron microscopy and transmission electron microscopy, Conclusion It is suggested from this study that serum-free culturing procedure can enrich cancer stem-like cells in nasopharyngeal carcinoma cell line CNE-2Z, and CD133 and CD44 can be considered as their special surface markers to be used as to identify nasopharyngeal cancer stem-like cells. Moreover, these cells show special ultrastructural features as well.

关 键 词：鼻咽癌 肿瘤干细胞样细胞 悬浮培养法 细胞鉴定 

分 类 号：R739.63[医药卫生—肿瘤]