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目的探讨不同培养条件下乳腺癌MCF-7细胞的分裂特点,建立快速、有效的乳腺癌干细胞富集方法。方法将MCF-7细胞分别采用完全培养基静置培养(A组)、完全培养基摇动培养(B组)、细胞因子静置培养(C组)和细胞因子摇动培养(D组)12、24、36 h,倒置相差显微镜下观察各组细胞分裂情况,计算克隆形成率,采用流式细胞术检测CD44+/CD24-/low细胞亚群的比例变化。结果 B组和C组培养12 h,棒状分裂细胞约占30%;24 h后分别约占50%和60%;36 h后形成大的细胞克隆。D组培养12 h,棒状分裂细胞约占50%;24 h后约占80%,可见多个细胞克隆;36 h后克隆细胞数减少。D组培养12 h,CD44+/CD24-/low细胞亚群比例(8.05%)为B组(0.99%)的8倍,C组(3.80%)的2.1倍;培养24 h,D组(15.24%)为B组(4.83%)的3倍,C组(2.30%)的6倍;培养36 h,D组CD44+/CD24-/low细胞亚群比例降至9.68%,约为B组(0.95%)和C组(1.03%)的9倍。结论在细胞因子摇动培养条件下,MCF-7细胞分裂加快,分裂象增多,细胞系中CD44+/CD24-/low亚群比例增加较快,干细胞池增加明显,表明无血清摇动悬浮培养为富集乳腺癌干细胞的快速、有效的方法。
OBJECTIVE: To investigate the characteristics of cleavage of breast cancer MCF-7 cells under different culture conditions and to establish a rapid and effective method for breast cancer stem cell enrichment. Methods MCF-7 cells were cultured in complete culture medium (group A), complete medium shaking culture (group B), cytokine resting culture (group C) and cytokine shake culture (group D) , 36 h. The cell division of each group was observed under inverted phase contrast microscope, and the formation rate of colony formation was calculated. The proportion of CD44 + / CD24- / low cell subsets was detected by flow cytometry. Results After cultured for 12 hours in group B and group C, about 30% of the cells were divided into rod-like cells. After 24 hours, they accounted for about 50% and 60% respectively. After 36 hours, large cell clones were formed. In group D, 12 hours after culture, about 50% of the cells were divided into rod-like cells. After 24 hours, about 80% of the cells were divided into cells, showing multiple cell clones. After 36 hours, the numbers of cloned cells decreased. In group D, the percentage of CD44 + / CD24- / low cell subpopulation (8.05%) was 8 times that of group B (0.99%) and 2.1 times of that of group C (3.80% ) Was 3 times of that of group B (4.83%) and 6 times of that of group C (2.30%). The proportion of CD44 + / CD24- / low cell subsets in group D decreased to 9.68% ) And 9 times of group C (1.03%). Conclusions Under the conditions of cytokine shake culture, the division of MCF-7 cells accelerates and the number of mitotic figures increases. The proportion of CD44 + / CD24- / low subpopulation in cell lines increases rapidly and the number of stem cell pools increases significantly, indicating that serum-free shake suspension culture is enrichment Breast cancer stem cells fast, effective method.