条件性重编程技术分离培养人原代乳腺癌干细胞

Isolation and Cultivation of Human Primary Breast Cancer Stem Cells Based on Conditionally Reprogrammed Cells Technique

目的应用条件性重编程细胞(Conditionally Reprogrammed Cells,CRCs)技术建立高效、稳定的人乳腺癌干细胞体外培养,建立潜在的乳腺癌干细胞靶向药研究模型。方法收集人乳腺癌组织样本,采用CRCs技术分离培养原代乳腺癌细胞,用CD24免疫磁珠标记原代乳腺癌细胞后使用磁性细胞分选仪分选出乳腺癌干细胞,免疫荧光法测定广谱细胞角蛋白(Pan Cytokeratin,pan-CK)表达,流式细胞术检测干细胞标志物CD44,CD24和乙醛脱氢酶1(acetaldehyde dehydrogenase,ALDH1)的表达水平,克隆形成实验和微球体形成实验分别用于测定克隆形成和乳腺微球体形成。结果利用CRCs技术分离培养的原代乳腺癌细胞能够稳定传代扩增至第9代,并保持乳腺癌细胞的特征。采用CRCs技术结合磁性细胞分选仪对乳腺癌原代细胞进行培养和分选后,乳腺癌非干性细胞微球体形成率显著低于干性细胞,乳腺癌非干性细胞克隆形成率显著低于干性细胞。乳腺癌干细胞可扩增至第5代。结论联合利用CRCs和免疫磁珠分选能够从原代乳腺癌组织中高效分离出具有肿瘤干细胞生物学特性并能稳定传代扩增的乳腺癌干细胞,这一体系可作为潜在的乳腺癌干细胞靶向药物研究模型。

OBJECTIVE To establish efficient and stable human breast cancer stem cells in vitro by conditionally reprogrammed ceus(CRCs)technique,and to establish potential breast cancer stem cells targeted drug research model.METHODS Primary breast cancer cells were isolated from breast cancer patients surgical specimens and cultured by CRCs technique,and breast cancer stem cells were then selected by magnetic cell sorter after primary breast cancer cells being labeled with CD24 immunomagnetic beads.The expression of Pan Cytokeratin(pan-CK)was detected by immunofluorescence.The expression levels of cancer stem cells biomarkers CD44,CD24,and ALDH1 was measured by flow cytometry.The cell clonies,and formation of mammospheres were determined by colony formation experiment,and mammospheres formation experiment.RESULTS The primary breast cancer cells isolated and cultivated by CRCs technique could be continuously cultivated in vitro for up to 9 passages,and the characteristics of breast cancer were maintained.After the primary breast cancer cells were cultured by CRCs technique and sorted by magnetic cell sorter,the mammosphere formation rate and clony formation rate of breast cancer non-stem cells were significantly lower than that of breast cancer stem cells.Breast cancer stem cells could be passaged to 5 passages.CONCLUSION Breast cancer stem cells can be isolated efficiently from primary breast cancer tissues and can be proliferated stably by the combined applicaiton of CRCs and magnetic cell sorter.And the system has a strong potential to be a new breast cancer stem cells targeted drug research model.