食管肿瘤干细胞抗辐射特性的实验研究

Radioresistance of esophageal cancer stem cells

目的 :探讨食管肿瘤干细胞的抗辐射特性。方法 :采用无血清培养基培养人食管癌细胞ECA109,分离食管肿瘤干细胞,MTT法检测不同浓度环氧化酶-2选择性抑制剂NS398(2.5、5.0、10.0、20.0、40.0、80.0μmol/L)对细胞增殖的抑制作用,克隆形成实验检测亲本细胞和细胞球的增敏效应,成球实验分析NS398联合X线照射对细胞成球能力的影响,Western blot检测细胞β-catenin蛋白的表达水平。结果:NS398对亲本细胞和细胞球的增殖抑制作用均具有时间、浓度依赖性。20μmol/L NS398作用后,亲本细胞Do、Dq和SF2值均减小(P<0.05),放射增敏比(sensitization enhancement ratio,SER)为1.17;20μmol/L NS398作用后,细胞球Do、SF2减小(P<0.05),SER为1.12。照射使ECA109细胞成球率增加(P<0.05)。NS398联合X线照射组与单纯照射组相比,细胞成球率显著降低(t=7.01,P<0.01),亲本细胞和细胞球β-catenin的表达水平均降低(t=10.15,P<0.01;t=3.225,P<0.05)。结论 :细胞球的增敏效应小于亲本细胞,具有抗辐射特性,其机制可能与抑制细胞β-catenin蛋白表达有关。

Objective：To study the radioresistance of esophageal cancer stem cells. Methods：The ECA109 cells were cultured in serum-free medium to enrich esophageal stem-like cells. MTT assay was performed to detect the multiplication capacity of cancer ceils treated with different concentrations of cyclooxygenase-2 （COX-2） selective inhibitor NS398 （2.5,5.0,10.0,20.0,40.0 and 80.0μmol/L）. Sensitization effects of parental cells and spheroid cells were evaluated by clone formation assay. Sphere-forming assay was aimed to analyze the effect of NS398 combined with X-ray radiation on cell spheres formation. Western blot was performed to determine the expression level of β-catenin. Results：NS398 inhibited the proliferations of parental cells and spheroid cells in a time-and concentration- dependent way. After treated with 20 p, mol/LNS398,Do,Dq and SF2 values of parental cells were all decreased （P 〈 0.05） ,and the sensitization enhancement ratio （SER） was 1.17. Do and SF2 values of spheroid cells were decreased （P 〈 0.05） ,and the SER was 1.12. Formation rate of the cell spheres was increased after irradiation （P 〈 0.05）. Compared with the radiation alone group,the formation rate of the cell spheres and β-catenin expression of parental cells and spheroid cells were decreased in the combination group （t = 7.01 ,P 〈 0.01 ;t = 10.15 ,P 〈 0.01 ;t = 3.225,P 〈 0.05）. Conclusion：The sensitization effect of spheroid cells is less than parental cells,which indicates that spheroid cells has the radioresistance. It may be correlated with the inhibition of the expression level of β-catenin.