人结肠癌细胞株CW-2干细胞富集方法的比较

Comparison on enriching methods for colon cancer stem cells from human colon cancer cell line CW-2

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;分别测量不同时间点(4、7、9、14、21、28d)各组小鼠成瘤情况;免疫组化检测移植瘤中CD44和EPCAM的表达情况。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+EPCAM+细胞,细胞比例分别为57.39%、73.85%、89.57%;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)、(4897.00±1154.15)、(11070.00±3559.86)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比较差异有统计学意义(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72h后穿过人工基底膜的细胞数分别为(98±7)/视野、(135±7)/视野、(188±6)/视野,3组之间差异有统计学意义(P<0.05);各组移植瘤中均表达CD44和EPCAM。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+奥沙利铂处理细胞、单纯无血清悬浮培养细胞。结论多重联合法富集肿瘤干细胞的效果明显强于单纯无血清悬浮培养和无血清悬浮培养+化疗药物法,是获得肿瘤干细胞较好的方法。

Objective To investigate an effective method to enrich tumor stem cells by comparing the efficiency of the 3 cell isolation methods.Methods CW-2 stem cells were enriched with 3 different methods,including simple serum-free medium,serum-free medium combination with oxaliplatin,and the second one further combined with flow cytometry （multi-combination）.The enrichment efficiency of the 3 methods was then analyzed and compared with flow cytometry,tumorigenicity assay in NOD-SCID mice and Transwell invasion assay in vitro.The expressions of CD44 and EPCAM were determined by immunohistochemistry method.Results The frequency of CD44＋ EPCAM＋ cells were 57.39%,73.85 % and 89.57% separately in cells enriched by simple serum-free medium,serum-free medium combination with oxaliplatin and multi-combination;The tumor xenograft of cells enriched by multi-combination were formed earliest and grew fastest than the tumor xenograft of cells enriched by pure serum-free medium and serum-free medium combination with oxaliplatin.The volume of transplantation tumor from cells enriched by multi-combination were （209.25±31.54）,（500.00±72.90）,（852.75±157.51）,（2 280.00±440.91）,（4 897.00±1 154.15）,（11 070.00±3 559.86） mm3 separately on the 4th,7th,9th,14th,21st and 28th day.Tumor-forming capability of the 3 group cells had statistically significant differences （P0.05）.The number of cells past through man-made GBM to down-room were （98±7） cells per field view,（135±7） cells per field of view,and （188±6） cells per field of view separately in cells enriched by simple serum-free medium,serum-free medium combination with oxaliplatin and multi-combination.Invasion capability of the 3 group cells had statistically significant differences （P0.05）.So the cells enriched by multi-combination had most tumor stem cells than the cells enriched by other 2 methods.CD44 and EPCAM all expressed in each group of tumor xenograft.Conclusion Multi-combination is highest than pure serum-free medium and serum-free medium combination with oxaliplatin in enrichment capability of tumor stem cells and is a better method to enrich tumor stem cells.