肺癌干细胞的球体形成与致瘤性分析  

The sphere formation and tumorigenesis of lung cancer stem cells

作　　者：耿沁[1] 董强刚[1] 姚明[2] 郑必强[1] 胡晶莹[3] 周瑾[1] 赵仰星[3] 闫明霞[2] 甘愉[3] 

机构地区：[1]上海交通大学上海市肿瘤研究所分子诊断实验室,上海200032 [2]上海交通大学上海市肿瘤研究所实验病理室,上海200032 [3]上海交通大学上海市肿瘤研究所免疫与分子生物学研究室,上海200032

出　　处：《肿瘤》2008年第9期751-754,共4页Tumor

摘　　要：目的：从人肺腺癌细胞株SPC—A1中诱导球体形成，对其中的肺癌干细胞进行鉴定并评估其致瘤能力。方法：无血清条件下培养肺癌细胞，采用表皮生长因子（epidermal growth factor，EGF）、重组人胰岛素样生长因子-1（insulin—like growth factor-1，IGF-1）和重组人成纤维细胞生长因子-10（fibroblast growth factor—10，FGF-10）等细胞生长因子诱导球体形成，球体细胞采用RT—PCR及免疫荧光技术检测干细胞相关标志的表达，并通过NOD—SCID小鼠移植评估其致瘤性。结果：SPC—A1肺腺癌细胞经培养5—10d后，即可形成悬浮生长的细胞球体（肺球体）。RT—PCR检测显示肺球体细胞表达肺癌干细胞表面标志CD24和CD221．细支气管肺泡干细胞标志Clara细胞分泌蛋白和肺泡活性蛋白C，胚胎干细胞主干基因OCT4、Nanog和Bmi-1，以及肺干细咆主干基因甲状腺转录因子-1。采用免疫荧光检测其中3个标志的蛋白表达，证实80％以上的肺球体细胞呈Clara细胞分泌蛋白、肺泡活性蛋白C和OCT4染色阳性。肺球体细胞具有高致瘤性，在NOD—SCID小鼠中移植5103个细胞即可形成肿瘤。结论：肺球体细胞中肺癌干细胞高度富集并具有致瘤性。Objective：To induce the sphere formation from human lung adenocarcinoma cell line SPC-A1, identify cancer stem cells among the spheres, and evaluate their tumorigenic potential. Methods：The spheres were induced by incubation of cancer cells in the serum-free medium. The sphere formation was induced by epidermal growth factor （EGF）, insulin-like growth factor-1 （IGF-1）, and flbroblast growth factor-10 （FGF-10）. The stem cell-related markers were measured by RT-PCR and immunofluorescent staining in the resuhant sphere-forming cells. Their tumorigenesis was tested in NOD/SCID mice. Results： The float-growing spheres （ named pulmospheres） were developed after SPC-A1 cells were cultured for 5-10 days. RT-PCR demonstrated that the pulmospheres expressed the lung cancer stem cell surface markers （CD24 and CD221 ） , the bronchioalveolar stem cell markers [ surfactant proteins C （SP-C） and Clara cell secretoIy protein （CCSP） ], the embryonic stem cell sternness genes （ OCT4, Nanog, and Bmi-1 ） , and the lung stem cell stemness gene （ TTF-1）. Among these markers, the expressions of SP-C, CCSP, and OCT4 were further evaluated with the immunofluorescent staining. Approximately 80 % of the sphere cells were shown to be positive for the proteins. The pulmosphere cells were highly tumorigenic in NOD-SCID mice, with tumor development after implantation of 5 × 10^3 cells per mouse. Conclusion： The lung cancer stem cells are enriched in the pulmosphere-forming cell population, which are tumorigenic.

关 键 词：腺癌 细支气管肺泡 细胞培养技术 干细胞 球体 动物实验 

分 类 号：R734.2[医药卫生—肿瘤]