肺癌细胞株A549的常见肿瘤干细胞抗原分析和细胞分选比较

Phenotypic analysis of common cancer stem cell markers and cell sorting in lung cancer cell line A549

背景:多项研究表明癌症有可能是起源于肿瘤组织内一个细胞亚群即肿瘤干细胞,近期的研究发现肺癌内也存在肿瘤干细胞,但肺癌内存在的肿瘤干细胞的表型目前仍未取得一致。目的:对人类非小细胞肺癌细胞株A549通过目前最常见的肿瘤干细胞表面抗原进行全面系统分析。方法:A549肺癌细胞株在常规高糖培养环境下,培养1周后进行表面抗原的检测。使用流式细胞术分析A549细胞CD133,CD44,CD24以及ABC运转蛋白家族G2的表达情况,并且通过流式分选的方法分离出CD44^+/CD24+和CD44^+/CD24^-两类亚群细胞,在低黏附、无血清并添加生长因子的环境下观察肿瘤干细胞肿瘤球形成的能力。结果与结论:(1)细胞表型变化:A549细胞中度表达肿瘤干细胞常见的抗原CD44(64.23%)和CD24(58.62%),而CD133和ABC运转蛋白家族G2的表达率很低,均约为0.9%,其中CD133/CD44双阳性的细胞数比例极低。表型为CD44^+/CD24+和CD44^+/CD24^-的细胞群比例分别为54.64%和23.38%。两类亚群细胞在碱性成纤维细胞生长因子及表皮生长因子存在的条件下,无血清培养均可形成肿瘤干细胞肿瘤球;(2)肿瘤球形成效率:CD44^+/CD24+和CD44^+/CD24^-的细胞群其肿瘤干细胞肿瘤球形成效率与A549细胞相比差异无显著性意义;(3)结果说明:CD44和CD24也许并非肺癌肿瘤干细胞的特异性标志,对于肺癌内肿瘤干细胞的表型鉴别还需要更多的研究。

BACKGROUND:It has been widely reported that the most types of cancer are probably originated from cancer stem cells(CSCs)which are subpopulations of tumor cells.Recent studies also suggested that lung cancer could arise from CSCs.However,the phenotypic characteristics of CSCs in lung cancer have not been precisely described.OBJECTIVE:To systematically analyze the expression of the most common CSC markers in cell line A549.METHODS:A549cells were cultured for1week under the condition of conventional high glucose.After that,flow cytometry was used to assess the expression of putative stem cell markers,including CD133,CD24,CD44and ABCG2.Cells were then sorted according to the expression of CD44and CD24markers by fluorescence-activated cell sorting(FACS)and characterized using their sphere-forming capacity in serum free medium supplemented with several growth factors.RESULTS AND CONCLUSION:A549cells expressed the CSC markers CD44and CD24at64.23%and58.62%,whereas the expression of both ABCG2and CD133was around0.9%.Double-positive CD44/133populations were rare.CD44+/CD24+and CD44+/CD24-subpopulations respectively exhibited54.64%and23.38%expression.CD44+/CD24+and CD44+/CD24-subsets were sorted by FACS.Both isolated subpopulations formed spheres in the serum free medium supplemented with basic fibroblast growth factor and epidermal growth factor.However,there was no significant difference in the sphere formation efficiency among CD44+/CD24+and CD44+/CD24-subsets as well as A549cells.Our findings suggest that CD44and CD24cannot be considered as potential markers for isolating lung CSCs in cell line A549,and further investigation using in vivo assays is required.