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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 compare the enrichment efficiency of three kinds of cell sorting methods on human colon cancer cell line CW-2 stem cells and to explore an effective method for obtaining cancer stem cells. Methods The human colon cancer cell line CW-2 stem cells were enriched by simple serum-free suspension culture, serum-free suspension culture with oxaliplatin, serum-free suspension culture and oxaliplatin + flow cytometry (multiple combination) Flow cytometry, NOD-SCID mouse tumorigenicity assay and Transwell invasion assay were used to compare the efficiency of the three methods. The tumorigenesis of mice in each group at different time points (4, 7, 9, 14, 21, 28d) Immunohistochemistry was used to detect the expression of CD44 and EPCAM in the xenografts. Results The percentage of CD44 + EPCAM + cells with colon cancer stem cells in serum-free suspension culture, serum-free suspension culture + oxaliplatin-treated cells and multiple combined sorted cells were 57.39%, 73.85%, 89.57% The NOX-SCID mice were co-sorted and the tumorigenic time was earlier and the tumor growth rate was faster. The volume of each tumor was (209.25 ± 31.54), (500.00 ± 72.90), (852.75 ± 157.51) and (2280.00 ± 440.91 ), (4897.00 ± 1154.15) and (11070.00 ± 3559.86) mm3, respectively. There was a significant difference in tumorigenicity between serum-free suspension culture and oxaliplatin-treated cells (P <0.05) Serum-free suspension culture cells, serum-free suspension culture + oxaliplatin-treated cells and multiple combined sorting cells after seeding 72h after passage through the artificial basement membrane cells were (98 ± 7) / field, (135 ± 7) / Field of vision, (188 ± 6) / field of vision. The difference between the three groups was statistically significant (P <0.05). CD44 and EPCAM were expressed in all the groups. The results showed that the highest concentration of stem cells in the cells obtained from the three sorts of sorting methods was: cells after multiple combined sorting, cells cultured in serum-free suspension culture + oxaliplatin and cultured in serum-free suspension alone. Conclusions Multi-combination method is more effective in enriching cancer stem cells than pure serum-free suspension culture and serum-free suspension culture + chemotherapy drugs, which is a good method to obtain tumor stem cells.