人结肠癌细胞株CW-2干细胞富集方法的比较.docVIP

人结肠癌细胞株CW-2干细胞富集方法的比较 刘 美，李少林，陈 宓，李小青，龙 浩 （400016 重庆，重庆医科大学基础医学院放射医学教研室） [摘要] 目的 比较3种细胞分选方法对人结肠癌细胞株CW-2干细胞的富集效率，探讨获得肿瘤干细胞的有效方法。方法 采用单纯无血清悬浮培养、无血清悬浮培养＋奥沙利铂、无血清悬浮培养＋奥沙利铂＋流式细胞术（多重联合）分别富集人结肠癌细胞株CW-2干细胞；应用流式细胞术、NOD-SCID小鼠致瘤实验和Transwell侵袭实验比较3种方法的富集效率。结果 无血清悬浮培养细胞、无血清悬浮培养＋奥沙利铂处理细胞和多重联合分选细胞中具有结肠癌干细胞特性的CD44[基金项目] 国家自然科学基金EPCAM[通信作者] 李少林，电话：（023E-mail: shaolinli202@细胞，分别为57.39%、73.85%、89.57%；分别测量不同时间点（4.、7、9、14、21、28 d）各组小鼠成瘤情况，多重联合分选细胞接种NOD-SCID小鼠后成瘤时间早、瘤体增长速度快，各时间点体积分别为（209.25±31.54）、（500.00±72.90）、（852.75±157.51）、（2280.00±440.91）、(4 897.00±1 154.15)、（11 070.00±3 559.86）mm3，与无血清悬浮培养细胞、无血清悬浮培养＋奥沙利铂处理细胞的成瘤能力差异有统计学意义（P ＜0.05）；单纯无血清悬浮培养细胞、无血清悬浮培养+奥沙利铂处理细胞和多重联合分选后细胞接种72 h后穿过人工基底膜的细胞数分别为（98±7）、（135±7）、（188±6）个/视野，3组之间差异有统计学意义（P ＜ 0.05）。故3种分选方法所获得细胞中干细胞含量由高到低为：多重联合分选后细胞、无血清悬浮培养 [基金项目] 国家自然科学基金 [通信作者] 李少林，电话：（023E-mail: shaolinli202@ [关键词] 肿瘤干细胞；结肠肿瘤；流式细胞术；细胞分离；细胞培养技术 [中图分类号] R735.3+5；R73-35+1；R73-35+2 [文献标志码] A Comparison of methods of the isolation colon cancer stem cells from human colon cancer cell line CW-2 Liu Mei, li Shaolin, Chen Mi, Li Xiaoqing, Long Hao (Department of Radiation Medicine, Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016) [Abstrat] Objective To investigate the effective method of enriching tumor stem cells by comparison the efficiency of three cell isolation methods. Methods CW-2 stem cells were enriched with three different methods which are simple serum-free medium, serum-free medium combination with oxaliplatin ，mult- combination;and then analyze and compare the enrichment efficiency of the three methods with flow cytometry, tumorigenicity assay in NOD-SCID mice and Transwell invasion assay in vitro. Results The frequencys of CD 44 + EPCAM + cells [1] were 57.39%、73.85 %、 89.57% separately in cells enriched by simple serum-free medium, serum-free medium combination with oxaliplatin and mult- combination;The tumor xenograft of cells enriched by multi-combination were fo