乳腺癌细胞系中肿瘤干细胞相关亚群的初步研究

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

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。

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.