中文摘要：

目的：探讨肿瘤微环境在乳腺癌干细胞（breastcancerstemcells，BCSCs）培养鉴定及分化过程中的影响及意义。方法：采用无血清培养液PCM-2及成纤维细胞上清液对乳腺癌细胞及MCF一7细胞进行原代培养。观察乳腺癌细胞微球体形成状况，MTT比色法检测乳腺癌细胞的增殖能力，免疫细胞化学方法检测乳腺癌干细胞标记物及上皮间质标记物的表达，并通过RT—PCR进行验证。结果：无血清培养液PCM一2培养的原代细胞微球体的直径大于成纤维细胞上清液的培养（t=4．996，P=0．002），且原代细胞中ALDHl（aldehyde dehydrogenase1）的表达率高于后者。成纤维细胞上清液培养的细胞生长速度较无血清培养液PCM一2快，差异具有统计学意义（P=0．004）。RT—PCR检测发现无血清培养液PCM一2培养的原代细胞中ALDHl表达上调，E—cadherin、Vimentin表达下调。结论：在乳腺癌原代细胞和MCF一7细胞中可以采用无血清悬浮培养方法富集BCSCs样微球体，成纤维细胞上清液能够促进BCSCs样微球体的增殖与分化。提示乳腺肿瘤微环境在乳腺癌细胞的生长增殖过程中发挥了至关重要的作用。

英文摘要：

Objective： To investigate the influence and significance of tumor microenvironment in breast cancer stem-cell culture and identification. Methods： Cells isolated from primary breast cancer tissues were cultured in vitro in a serum-free medium PCM-2 and in the supernatant of cultured fibroblasts. The MCF-7 breast cancer cell line was used as the control group. The status of the micro- spheres was observed, and the proliferative capacity of the cells was detected by methyl thiazolyl tetrazolium assay. The expression of stem cell and epithelial-mesenchymal markers were detected by real-time reverse transcription polymerase chain reaction. Results： The diameter of microspheres in PCM-2 gradually increased with prolonged incubation time （t=4.996, P=0.002）. The cells in the supema- tant of cultured fibroblasts increased daily and mostly exhibited a spindle cell growth. The growth rate of primary breast cancer cells was faster in the supernatant of cultured fibroblasts than in PCM-2 （P=0.004）. Compared with the case of cells in the supernatant of cul- tured fibroblasts, aldehyde dehydrogenase 1 was upregulated in the primary breast cancer cells cultured in serum-free medium PCM-2, whereas E-cadherin and Vimentin were downregulated. Conclusion： Serum-free culture can be one of the best methods for enriching breast cancer stem cell-like mammospheres. The tumor micro-environment serves a vital function in the growth and development of tu- mor ceils and in the evolution of breast cancer.