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

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

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、1.0μg/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1.0μg/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。

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.