目的从人肺癌细胞系A549中分离并鉴定肺癌干细胞。方法将A549细胞置于无血清条件培养基中培养,形成细胞球,采用平皿克隆形成实验、MTT细胞增殖实验、RT-PCR、Western blot、免疫荧光技术,检测并比较A549细胞和A549细胞球的增殖能力及干细胞相关标记的表达水平,鉴定细胞球的肿瘤干细胞特性。结果利用无血清条件培养基从A549细胞中分离出肿瘤干细胞样细胞;相比A549细胞,细胞球呈悬浮生长,增殖能力和克隆形成数目(104±7)高于贴壁细胞[(37±3)(P<0.05)],且干细胞相关标记Sox2(2.68±0.39)和Oct4(1.23±0.12)表达水平明显提高[贴壁细胞Sox2(0.05±0.02)、Oct4(0.10±0.02),P<0.05]。结论运用无血清条件培养基可以从A549细胞系中有效富集肺癌干细胞样细胞。 Objective To isolate and identify lung cancer stem cells from human lung cancer cell line A549. Methods A549 cells were cultured in serum-free conditioned medium to form cell spheres. The proliferation of A549 cells and A549 cells were detected and compared by using plate clone formation assay, MTT cell proliferation assay, RT-PCR, Western blot and immunofluorescence techniques Proliferation ability and expression level of related markers of stem cells to identify the characteristics of tumor stem cells of the cell sphere. Results The tumor stem cell-like cells were isolated from A549 cells with serum-free conditioned medium. Compared with A549 cells, the cell spheres grew in suspension and the proliferation and clonogenic capacity (104 ± 7) were higher than that of adherent cells [(37 ± 3) (P <0.05)]. The expression of Sox2 (2.68 ± 0.39) and Oct4 (1.23 ± 0.12) in stem cells were significantly increased ]. Conclusions Lung cancer stem cell-like cells can be effectively enriched from A549 cell line using serum-free conditioned medium.