上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (6): 736-.doi: 10.3969/j.issn.1674-8115.2012.06.010

• 论著（基础研究） • 上一篇    下一篇

CD133+A549肺癌细胞的分选及其肿瘤干细胞和间质化相关基因的表达

任涟萍, 王 佳, 郭雪君   

  1. 上海交通大学 |医学院附属新华医院呼吸内科, 上海 200092
  • 出版日期:2012-06-28 发布日期:2012-07-02
  • 通讯作者: 郭雪君, 电子信箱: guoxjz@yahoo.com.cn。
  • 作者简介:任涟萍（1971—）, 女, 副主任医师, 硕士；电子信箱: ren_lianping@yahoo.com.cn。

Isolation and characteristics of cancer stemness and epithelial-mesenchymal transition of CD133+A549 lung cancer cells

REN Lian-ping, WANG Jia, GUO Xue-jun   

  1. Department of Respiratory Medicine, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
  • Online:2012-06-28 Published:2012-07-02

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摘要：

目的 分离CD133+A549细胞并对其肿瘤干细胞和上皮间质转变（EMT）特性进行初步研究。方法 采用免疫磁珠法分离并鉴定A549细胞，Real-Time PCR法和Western blotting法鉴定分离的细胞。Real-Time PCR法测定CD133+A549和CD133－A549细胞中肿瘤干细胞标志物（CXCR4、Oct4、CD44、Bmi1、EpCam）和EMT标志物（E-Cadherin和Zeb1）mRNA的表达。分选后的CD133+A549和CD133－A549细胞经不同浓度（0、0.25、0.5、1.0 μg/mL）阿霉素处理72 h，采用细胞增殖实验检测细胞的相对存活率。结果 经CD133微小磁珠分离后，成功获得CD133+A549和CD133－A549细胞，CD133+A549细胞的CD133表达明显高于CD133－A549细胞。Real-Time PCR检测结果显示，CD133+A549细胞中Oct4、CD44、Bmi1、EpCam mRNA的相对表达量均显著高于CD133－A549细胞[（1±0.16）vs（0.66±0.12）、（1±0.07）vs（0.48±0.04）、（1±0.03）vs（0.49±0.06）以及（1±0.14）vs（0.38±0.12）（P<0.05）]，但CXCR4 mRNA的相对表达量比较差异无统计学意义[（1±0.13）vs（0.73±0.14），P>0.05]； CD133+A549细胞中Zeb1 mRNA的相对表达量显著高于CD133－A549细胞[（1±0.09）vs（0.39±0.05），P<0.05]，但E-Cadherin mRNA的相对表达量显著低于CD133－A549细胞[（1±0.02）vs（4.98±0.04），P<0.05]。细胞增殖实验结果显示，采用0.5和1.0 μg/mL阿霉素处理后，CD133+A549细胞的相对存活率均显著高于CD133－A549细胞，差异有统计学意义[（85±9.4）% vs （67±13.1）%，P<0.05；（80±14.9）% vs（56±6.3）%，P<0.01]。结论 采用免疫磁珠分离法可成功分离CD133+A549和CD133－A549细胞；CD133+ A549细胞中有其他肿瘤干细胞标志物的表达，并表现出EMT特性。CD133可能是肺癌肿瘤干细胞和EMT特征的标志物。

关键词: CD133, 肿瘤干细胞, 上皮间质转变, 肺癌

Abstract:

Objective To isolate CD133+A549 cells, and investigate the characteristics of cancer stemness and epithelial-mesenchymal transition (EMT). Methods A549 cells were isolated by immunomagnetic beads method, and were identified by Real-Time PCR and Western blotting. The expression of cancer stem cell markers (CXCR4, Oct4, CD44, Bmi1 and EpCam) and EMT markers (E-Cadherin and Zeb1) mRNA in CD133+A549 cells and CD133－A549 cells was detected by Real-Time PCR. The isolated CD133+A549 cells and CD133－A549 cells were treated with different concentrations (0, 0.25, 0.5 and 1.0 μg/mL) of adriamycin for 72 h, and the relative survival rates of cells were determined by cell viability assay. Results After isolation through CD133 antibody conjugated microbeads, CD133+ A549 cells and CD133－A549 cells were successfully obtained, and the expression of CD133 in CD133+A549 cells was significantly higher than that in CD133－A549 cells. Real-Time PCR revealed that the relative expression of Oct4, CD44, Bmi1 and EpCam mRNA in CD133+A549 cells was significantly higher than that in CD133－A549 cells [ (1±0.16) vs（0.66±0.12）,（1±0.07） vs （0.48±0.04）,（1±0.03） vs （0.49±0.06）, and (1±0.14） vs (0.38±0.12）; P<0.05], while there was no significant difference in the relative expression of CXCR4 mRNA between them [(1±0.13） vs (0.73±0.14），P>0.05]. The relative expression of Zeb1 mRNA in CD133+A549 cells was significantly higher than that in CD133－A549 cells [(1±0.09) vs (0.39±0.05），P<0.05], while the relative expression of E-Cadherin mRNA in CD133+A549 cells was significantly lower than that in CD133－A549 cells [(1±0.02) vs (4.98±0.04），P<0.05]. Cell viability assay indicated that the relative survival rate of CD133+A549 cells was significantly higher than that in CD133－A549 cells after treatment with 0.5 and 1.0 μg/mL adriamycin [(85±9.4）% vs (67±13.1）%，P<0.05；(80±14.9）% vs (56±6.3）%，P<0.01]. Conclusion CD133+ A549 cells and CD133－A549 cells can be successfully isolated by immunomagnetic beads method. There is expression of other cancer stem cell markers in CD133+A549 cells, which exhibit characteristics of EMT. CD133 may be a potential biomarker of cancer stemness and EMT in lung cancer.

Key words: CD133, cancer stem cells, epithelial-mesenchymal transition, lung cancer