人肺腺癌细胞株SPC-A1诱导球体形成及肺癌干细胞致瘤能力评估

摘要

背景：目前关于肺癌干细胞是否可以形成球体及其致瘤能力尚缺乏明确的定论。目的：观察人肺腺癌细胞株SPC-A1诱导球体形成及肺癌干细胞的致瘤能力。方法：利用无血清-DF12培养液培养增殖期肺癌细胞株SPC-A1，加入重组人胰岛素样生长因子1和重组人表皮生长因子及重组人成纤维细胞生长因子10诱导球体形成，获得球体细胞沉淀物，进行免疫荧光检测和PCR 扩增，了解干细胞相关标志的表达情况。将肺球体细胞植入 NOD-SCID 免疫缺陷小鼠皮下，观察肿瘤生长情况，并利用活体荧光成像仪进行摄片。结果与结论：培养肺腺癌细胞SPC-A15-10 d获得肺球体。RT-PCR检测发现，肺球体细胞表达肺癌干细胞及细支气管肺泡干细胞标志物，CD24、CD221、CCSP和SP-C；另外，肺球体细胞与纯化的CD24+、CD221+肺癌干细胞同样表达肺干细胞的主干基因TTF-1、胚胎干细胞主干基因OCT4、Nanog和Bmi-1肺干细胞的主干基因TTF-1。荧光检测显示，80%以上的肺球体细胞中表达CCSP和OCT4；SPC-A1细胞具有肺泡Ⅱ型细胞特征，并表达SP-C蛋白，但仅约5%的细胞表达CCSP和OCT4。肺球体细胞皮下植入50 d，活体荧光成像可清晰显示小鼠体内肿瘤直径达1 cm。表明人肺腺癌细胞株SPC-A1可诱导球体形成，球体中富含肺癌干细胞并具有致瘤能力。%BACKGROUND:There is no clear conclusion on whether the lung cancer stem cels can induce to spheroid formation and have tumorigenicity. OBJECTIVE: To observe the spheroid formation induced by human lung adenocarcinoma cel line SPC-A1 and the tumorigenic ability of lung cancer stem cels. METHODS:SPC-A1 at proliferating phase was cultured in serum-free DF12 culture medium, and then recombinant human insulin-like growth factor-1, recombinant human epidermal growth factor, and recombinant human fibroblast growth factor-10 were added to induce spheroid cels. Immune fluorescence detection and PCR amplification were done to understand the expression of stem cel associated markers. NOD-SCID immunodeficient mice were subcutaneously implanted with lung spheroid cels to observe the tumor growth.In vivo fluorescence imager was used for radiography. RESULTS AND CONCLUSION:After 5-10 days, lung spheroid cels were harvested. RT-PCR results showed that lung spheroid cels were positive for CD24, CD221, CCSP and SP-C. In addition, the lung spheroid cels and purified CD24+, CD221+ lung cancer stem cels were both positive for TTF-1 of lung stem cels, OCT4 and Nanog of embryonic stem cels and TTF-1 of Bmi-1 lung stem cels. The fluorescence detection showed that over 80% lung spheroid cels expressed CCSP and OCT4; SPC-A1 cels had the characteristics of alveolar type II cels, and also expressed SP-C protein, but only about 5% of the cels expressed CCSP and OCT4. At 50 days after subcutaneous implantation of lung spheroid cels, in vivo fluorescence imaging showed that the diameter of tumor in mice was 1 cm, indicating human lung adenocarcinoma cel line SPC-A1 can induce the spheroid formation, and lung cancer stem cels rich in the cel spheres have the tumorigenic ability.