Preliminary Study of Maintaining in Floating-culture System and Screening Side Population in Human Breast Cancer Cell Line MCF-7
论文导师 王水,论文学位 硕士,论文专业 普通外科
论文单位 南京医科大学,点击次数 57,论文页数 52页File Size3460K
MCF-7 human breast cancer cell line;;breast cancer stem cell;; breast cancer sphere;; floating-culture system;; serum-free culture medium
目的研究无血清培养基悬浮培养MCF-7乳腺癌细胞系，筛选并鉴定MCF-7乳腺癌细胞系中的肿瘤干细胞相关亚群。 方法应用乳腺癌培养基在无血清的条件下悬浮培养MCF-7乳腺癌细胞系。通过无血清培养筛选乳腺癌细胞系肿瘤干细胞相关亚群，将其接种于含血清培养基，观察分化。应用单克隆形成实验、表面标志检测、HOECHST33342染色检测来确定培养出的细胞中肿瘤干细胞的比例及其培养后肿瘤干细胞含量的变化。 结果乳腺癌MCF-7细胞系中有约2.12％的肿瘤细胞在无血清培养基中能够存活、增殖，形成自由漂浮的细胞球。细胞球可连续传代，若重新接种于含血清培养基中可重新贴壁分化，贴壁分化后细胞形态与直接在含血清培养基中培养的MCF-7无明显差别。流式细胞仪表面标志检测，细胞球中约含83.13％表达CD44~+CD24~(-／LOW)的细胞，HOECHST33342染色提示细胞球中约含10.06％的Side Population细胞。 结论MCF-7细胞系可在含生长因子的无血清培养基中悬浮生长，并维持细胞系。该细胞系中含有比例较低的具有增殖和分化能力的乳腺癌干细胞相关亚群。表面标志以及HOECHST33342检测差异提示细胞球中仅约1／8细胞具有干细胞的功能。
Objectives: To study whether the human breast cancer line MCF-7 contains breast tumor stem cell and its growing patten in serum-free medium supplemented with mitogens. Methods: Adherent MCF-7 cells in serum-supplemented medium were dissociated to single cell suspensions and were seeded in serum-free medium supplemented with bFGF and EGF in 6-well plates. After the MCF-7 breast cancer spheres formed,they were dissociated and passaged periodically. Limiting dilution analysis and monoclonal formation assay were also performed. Antibody CD24~(-/LOW) CD44~+ cells and side population were detected with flow-cytometric analysis. MCF-7 breast cancer spheres were induced to differentiate in the medium with 10% FBS supplemented. Results: About 2.12% of MCF-7 cells are clonogenic in MCF-7 cell line. They can generate breast cancer spheres for prolonged times. MCF-7 spheres can be induced to differentiate and adherent to the bottom of the culture plates. The growing patterns of MCF-7 cell can be concerted by changing the culture mediums. By using a FACScan, there are 83.13% CD24~(-/LOW) CD44~+ cells and 20% sp cell in MCF-7 cell line. Conclusion: MCF-7cell line contains some breast cancer stem cells. The cell line can be maintained in serum-free mediumwith floating-culture method for prolonged times. There are 1/8 cells which are real breast cancer stem cells, according to the result of low-cytometric analysis.