目的：从人肺腺癌细胞系A549及H1299中分离富集含肿瘤干细胞的细胞球并鉴定其生物学特性。方法：用无血清悬浮培养的方法从人肺腺癌A549及H1299细胞株中富集得到肿瘤细胞球。将肿瘤细胞球传代扩增，体外利用CCK-8法、平皿克隆以及Transwell小室实验，研究细胞球的增殖情况、自我更新和侵袭转移能力；通过RT-PCR检测干细胞特异性转录因子Oct4、Nanog基因表达情况；体内利用裸鼠移植瘤形成实验研究肺癌细胞球的成瘤能力。鉴定细胞球的肿瘤干细胞特性。结果：在无血清悬浮培养下，A549及H1299细胞株3~6天后能形成稳定传代的肿瘤悬浮球，悬浮球的体外自我更新、克隆形成和侵袭转移等能力均高于其亲本细胞（P<0.05）；干细胞核心基因Oct4和Nanog的mRNA表达水平明显升高（P<0.05）；A549悬浮球可以明显提高裸鼠体内成瘤能力。结论：通过无血清悬浮培养法可有效富集A549及H1299细胞系中的干细胞成分，该法可成为快速易行构建肺腺癌干细胞模型的方法。%Objective:To isolate cell spheres containing cancer stem cells (CSCs) from lung cancer cell lines (A549 and H1299) and iden-tify their biological characteristics. Methods:By adopting the method of serum-free suspension culture, A549 cells and H1299 cells were cultured on no-adhesion plate to form tumor spheres. Clone assay, CCK-8 assay, and Transwell assay were employed to observe proliferation, self-renewal and invasion of tumor spheres. Besides, RT-PCR was performed to compare the expression levels of stem cell markers between sphere cells and adherent cells. Adherent A549 cells and A549 cell spheres were inoculated subcutaneously in nude mice and the tumor growth was assessed. Results:Isolated CSCs from A549 cells and H1299 cells in serum-free medium (SFM) without adhesion could grow as floating cell spheres. The results demonstrated that the self-renewal, proliferation and invasion of A549 and H1299 sphere cells were stronger than parent cells (P<0.05). When compared with adherent cells, the mRNA level of expres-sion of cell spheres' stem cell markers (Oct4 and Nanog) were significantly high (P<0.05) and A549 spheres had a stronger tumorigenici-ty in nude mice. Conclusion:SFM without adhesion can be a quick and easy method to construct stem cells model from human lung adenocarcinoma cell lines A549 and H1299.
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