乳腺癌细胞系中肿瘤干细胞相关亚群的初步研究

目的 通过乳腺癌细胞系及其干细胞的培养,化疗药干预和流式细胞仪筛选鉴定,探讨不同乳腺癌细胞系中CD44⁺CD24^-/low细胞比例,及富集乳腺癌干细胞相关亚群的方法。方法 通过细胞培养乳腺癌细胞系MCF-7、MDA-MB-231,观察生长曲线,比较化疗药物干预下生长情况;利用流式细胞仪检测两种乳腺癌细胞系中CD44⁺CD24^-/low细胞比例;无血清悬浮培养,化疗药(多西紫杉醇、表阿霉素)干预这两种乳腺癌细胞系,观察其是否形成细胞球。结果 (1)MDA-MB-231细胞系倍增时间短,生长速率高于MCF-7细胞系;(2)MCF-7细胞系中可能存在较大比例肿瘤干细胞,其对化疗抵制,能自我更新;(3)化疗敏感性用两独立样本t检验,MCF-7细胞,差异没有统计学意义;MDA-MB-231细胞,差异有统计学意义,提示MDA-MB-231细胞系对该方案化疗较敏感;(4)无血清悬浮培养,MDA-MB-231细胞系未发现明显细胞球;MCF-7细胞系初次无血清培养约6天出现细胞球。加入化疗药筛选后两种细胞,见大部分肿瘤细胞逐渐死亡,未发现明显细胞球;(5)流式细胞仪检测,MCF-7、MDA-MB-231两种细胞系中主要是CD44⁺CD24⁺亚群和CD44^-CD24⁺亚群,CD44⁺CD24^-/low细胞比例分别2.07%和0.20%。结论 (1)MDA-MB-231细胞系增值较快,恶性度相对较高,其对TA联合化疗药物较敏感;MCF-7细胞系中可能存在少量肿瘤干细胞,对化疗抵制,能自我更新;(2)无血清培养液培养MCF-7细胞系能形成悬浮细胞球;流式细胞仪检测两种细胞系中CD44⁺CD24^-/low细胞比例小;(3)CD44⁺CD24^-/low表型可能不是乳腺癌干细胞唯一特异性的表面标志。

Preliminary study on cancer Stem - like cells in human breast cancer cell Iines

Objective Under culturing different human breast cancer cell lines and the related content of cancer stem - like cells, we discuse CD44 ＋CD24 -/low cell proportion in breast cancer cell lines and related subsets enrichment method of breast cancer stem - like cells through effective intervention of the commonly chemotherapy and flow cytometric analysis. Methods Through the cell culture of breast cancer cell line＂ MCF - 7, MDA - MB -231 ＂, we observed the growth curve and compared grow under conditions with chemotherapy drugs intervention, we detected the expression proportion of CD44＋ CD24 -/low cell in the two breast cancer cell lines by flow cytometry. Under suspension culturing the above cell lines in serum - free medium, we observed clonal nonadherent spherical clusters by combining with or without chemotherapy scheme of TA scheme （ docetaxel ＋ table doxorubicin）. Results 1. The multiplial time of MDA - MB - 231 ceils was shorter, and its growth rate was higher than MCF - 7 cells. 2. The MCF - 7 cells might exist the larger proportion of cancer stem - like cells with chemotherapy resistance and self - update. 3. Chemotherapy sensitivity with two independent sample t - test, the result in MCF- 7 cells suggested no statistical significance（ P 〉 0.05 ）. The result in MDA -MB -231 cells showed obvious positive significance （P 〈 0.05 ）. MDA- MB- 231 cells were more sensitive to chemotherapy from them. 4. Under serum- free suspension cultures of two breast cell lines, MDA- MB -231 cells got no clear cell balls. MCF - 7 cells were found cell balls at the first 6 day. With chemotherapy, the majority of the two tumor cells gradually died, and there were no living cell balls. 5. Measured the content of CD44 ＋ CD24-/low cytometry cells by flow cytometry, the two above cell lines mainly contain CD44 ＋ CD24 ＋ subsets （74.2% , 95.7% ） and CD44 - CD24 ＋ subsets（23.3% ,3.95% ）. The CD44 ＋ CD24 -/low proportion of MCF - 7 and MDA - MB - 231 was 2.07% and 0.20%, respectirely. Conclusion 1. The MDA - MB - 231 cells line which growed faster had higher malignant degree and showed more sensitive than MCF - 7 cells line to TA chemotherapy scheme. 2. Under serum -free suspension cultures, MCF -7 cells grow clonal monadherent spherical clusters, and the two lines had small proportion of CD44 ＋ CD24 -/low cell with flow cytometric detection. 3. The CD44 ＋ CD24 -/low phenotype may not be the only specific surface markers of breast cancer stem cells.