乳腺癌新辅助化疗后的癌干细胞分离培养-第三军医大学学报.DOCVIP

乳腺癌新辅助化疗后的癌干细胞分离培养 董华英，吴诚义，陈元文（400016 重庆，重庆医科大学附属第一医院内分泌外科） [摘要] 目的：从不同周期新辅助化疗后的乳腺癌组织中分离培养乳腺癌干细胞(breast cancer stem cells, BCSCs)，观察其体外增殖和分化特性，研究新辅助化疗对BCSCs分离培养的影响。方法：在添加生长因子的无血清培养基（serum free media, SFM）中悬浮培养细胞获得富含BCSCs的微球体（mammospheres, MSs），通过克隆形成实验研究微球体细胞（mammospheres-derived cells, MSDCs）的自我更新能力；在添加5%胎牛血清的培养基中培养MSDCs诱导其分化，研究其分化能力；免疫细胞化学检测ESA和Nestin在MSDCs中的表达；流式细胞术（Flow Cytometry, FCM）ALDH1+细胞含量。结果：化疗1个周期的5例乳腺癌标本经培养均无MSs形成，化疗2个周期的6例标本经培养其中3例有MSs形成，化疗3个周期以上的9例标本中的5例可以直接分离出MSs，其余4例仅得到少量坏死的细胞和组织碎片；MSDCs在含血清的培养基中贴壁分化；MSDCs均含有ALDH1+细胞。结论：从新辅助化疗后的乳腺癌组织中分离BCSCs宜选取化疗两个周期后的标本；新辅助化疗可以富集BCSCs；ALDH1+表型的BCSCs比CD44+CD24-表型的BCSCs在乳腺癌中分布更加广泛。 [关键词] 乳腺肿瘤；化疗；肿瘤干细胞；细胞分离；细胞培养 Isolation and culture of breast cancer stem cells derived from breast cancer after neoadjuvant chemotherapy Dong Huaying, Wu Chengyi, Chen Yuanwen (Department of Endocrine Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China) [Abstract] Objective: To isolate and culture of breast cancer stem cells (BCSCs) from breast cancer tissues after different cycles of neoadjuvant chemotherapy, and investigate their proliferation and differentiation biological characteristics in vitro in order to obtain the impact of neoadjuvant chemotherapy on the isolation and culture of BCSCs. Methods: Breast tissue samples from 20 patients receiving TAC neoadjuvant chemotherapy in our department from October 2009 to April 2010 were collected. There were 5 cases with 1 cycle of chemotherapy, 6 cases with 2 cycles, 7 cases with 3 cycles, and 2 cases with 4 cycles. The tissue samples (1 cm×1 cm×1 cm) were obtained through operation and cut into pieces for culture. The suspended cells were cultured in serum-free media (SFM) supplemented with growth factors. Cells grown in these conditions as nonadherent spherical clusters of cells (usually named “mammospheres”, MSs) were enriched in BCSCs. The self-renewal capacity of mammospheres-derived cells (MSDCs) was evaluated by colony formation assay. Conver