人A549肺腺癌始动细胞的富集和鉴定﹡.doc

人A549肺腺癌始动细胞的富集和鉴定﹡ 林盛，张振华，饶明月，吴敬波△ （泸州医学院附属医院肿瘤科， 646000） 摘要:目的 利用紫杉醇联合无血清培养对A549肺腺癌始动细胞富集并鉴定富集亚群的干细胞特性。方法 对数生长期的A549细胞经胰酶消化，干细胞培养基重悬，得到成球状生长的细胞；传至第二代时加入紫杉醇作用48h，离心去除死细胞和紫杉醇，换新鲜干细胞培养基培养，至存活细胞恢复克隆生长后鉴定其干细胞相关特性。结果 用紫杉醇联合无血清培养法成功从A549细胞中富集得到的细胞高表达CD133/CD326分子及干细胞相关基因，具有多向分化潜能和更强的致瘤能力，具备干细胞相关特性。结论 紫杉醇联合无血清培养富集得到高表达CD133/CD326分子的细胞亚群，该亚群具备干细胞相关特性，可用于后续肺腺癌干细胞生物学特性的研究。 关键词:肿瘤始动细胞；A549;紫杉醇 Enrichment and identification of lung adenocarcinoma initiating cells from A549﹡ LIN Sheng, RAO Ming-yue, WU Jing-bo△ (Oncology Department, Affiliated Hospital of Luzhou Medical College, Sichuan 646000, China) Abstract: Objective To obtain the lung adenocarcinoma initiating cells from the A549 cell line based on paclitaxel treatment combination with serum-free cultivation and to validate spared cells can represent tumor initiating cells (TICs). Methods After dissociated by trypsogen, about 106/ml cells were suspended in serum-free medium supplemented with 0.4% BSA, insulin, basic fibroblast growth factor (bFGF), human recombinant epidermal growth factor (EGF) and obtained spheroid cells. At the second passage, paclitaxel was added at a concentration of 100nmol/L for 48 h and then replaced with completely fresh medium once or twice per week until new spheroids emerged. Results The subpopulation of cells that survived serum-free cultivation and paclitaxel treatment could highly express the CD133/CD326 molecular markers and have features of stemness including differentiation, high expression of CSCs-associated genes and stronger capability of tumorigenesis. Conclusions The survived subpopulation that highly express the CD133/CD326 molecular markers presenting the characteristics of stemness in vitro and in vivo, and could be used in future ＊基金项目：国家自然科学基金资助项目。△通讯作者，Email:wjb6147@163.com researches of biological functions. Key words: tumor initiating cells; A549; paclitaxel 非小细胞肺癌（NSCLC）是重要的恶性肿瘤致死原因。近十年以来，随着诊断水平的提高和综合治疗模式（新辅助化疗，高精度放疗以及分子靶向治疗）的改进，使肺癌病员有所受益，但总5年生存率改善并不明显。大量的研