目的：探讨以氟尿嘧啶（5-fluorouracil, 5-FU）处理并通过荷瘤小鼠模型体内传代的方法富集乳腺癌干细胞样细胞的可行性，为靶向肿瘤干细胞治疗奠定基础。方法：以乳腺癌细胞株4T1皮下接种小鼠制备荷瘤模型，以一定剂量5-FU腹腔注射4周；处死小鼠后取肿瘤组织制成细胞悬液，并接种小鼠制备下一代小鼠荷瘤模型，5-FU处理及再次传代方法同上，共传4代。对照组小鼠给予生理盐水注射，其余处理同模型组。流式细胞术检测各代肿瘤组织中CD44+CD24-/low细胞比例，Hoechast 33342染色法检测侧群（side population，SP）细胞的比例，免疫组化法检测CD55和ALDH1蛋白的表达，倒置显微镜观察乳腺癌细胞微球体的形成，小鼠致瘤实验检测不同肿瘤细胞的致瘤能力。结果：各代对照组小鼠模型肿瘤组织中CD44+CD24-/low细胞比例为（11.5±0.9）%，SP细胞比例为（9.7±1.3）%，ALDH1表达阴性，CD55强阳性表达细胞数为（0.6±03）%，乳腺癌细胞微球体比例为（0.5±0.2）%；5-FU处理组4代小鼠模型肿瘤组织中CD44+CD24-/low细胞比例分别为（49.8±1.2）%、（56.8%±1.7）%、（66.4±1.5）%、（69.0±1.6）%，SP细胞比例分别为（25.0±1.2）%、（42.6±2.8）%、（58.4±2.1）%、（61.3±2.6）%，ALDH1阳性表达细胞比例为（3.8±0.7）%、（14.1±2.4）%、（25.2±3.1）%、（27.5±27）%，CD55强阳性细胞比例为（7.8±1.6）%、（10.1±20）%、（15.6±1.4）%、（17.3±1.9）%，乳腺癌细胞微球体形成比例为（5.9±0.4）%；两组各相应指标之间差异均具有统计学意义（P<0.05或P<0.01）。5-FU作用后富集了肿瘤干细胞的第3代肿瘤细胞的致瘤作用显著强于对照组细胞(P<0.05)。结论：5-FU作用并通过荷瘤小鼠体内传代能够富集小鼠乳腺癌4T1细胞株中的肿瘤干细胞样细胞。

Objective:To explore the possibility of enriching breast cancer stem cell-like cells by cell passage in a tumor-bearing mouse model treated with 5-fluorouracil （5-FU） in vivo and to lay a foundation for cancer stem cell-targeted therapy. Methods: To establish a tumor-bearing model by inoculating breast cancer cell line 4T1 into mouse subcutaneously. 5-FU was injected into these mice intraperitonealy according to different groups. The mice were executed 4 weeks after injection. Cell suspension made from mouse tumor was injected into the mice again to establish the next generation of tumor-bearing mouse model. The mice were treated with 5-FU in the third and fourth generation as above. Four generations of the mouse model were established in all. The mice in control group were injected with normal saline instead of 5-FU, and the following four generation tumor-bearing models were treated as 5-FU group. Flow cytometry was used to measure the proportion of CD44+CD24-/low cells in different tumor tissues; Hoechast 33342 staining was used to measure the proportion of SP (side population) cells; and expressions of ALDH1 and CD55 proteins were detected by immunohistochemistry. The formation of mammospheres was observed under an inverted microscope. Tumorigenic ability of different tumor cells was detected by mouse tumorigenesis experiment. Results: In the control group, the proportion of CD44+CD24-/lowcells in mouse tumor was (11.5±0.9)%, and the proportion of SP cells was (9.7±13)%. The expression level of ALDH1 was negative, the proportion of cells in which CD55 was highly expressed, was (0.6±0.3)%, and the proportion of mammospheres was (0.5±0.2)%. While in 5-FU treated group, the proportions of CD44+CD24-/lowcells were respectively (49.8±1.2)%, (56.8±1.7)%, (66.4±1.5)% and (69.0±1.6)%; the proportions of SP cells were respectively (25.0±1.2)%, (42.6±2.8)%, (58.4±2.1)% and (61.3±2.6)%; the proportions of cells in which ALDH1 was positively expressed, were respectively (3.8±0.7)%, (14.1±24)%, (25.2±31)% and (275±2.7)%, and the proportions of cells in which CD55 were highly expressed, were respectively (7.8±16)%, (10.1±2.0)%, (15.6±1.4)% and (17.3±1.9)%, and the proportion of mammospheres was (5.9±04)%. The significant differences were found between the two groups（P<0.05 or P<0.01）. The third generation tumor cells enriching cancer stem cells were more tumorigenic than the control tumor cells. Conclusion: 5-FU enrichs the cancer stem cell-like cells in mouse breast cancer cell line 4T1 by the cell passage in the tumor-bearing mouse model in vivo.

河北省自然科学基金资助项目（No. 2012206135）；邢台市科技计划资助项目（No. 2012-ZC-085）