食管癌干细胞辐射抗性与相关蛋白表达的研究

Studies on the radiation resistance of esophageal cancer stem cells and the expressions of relative proteins

目的探讨NS398联合x射线照射对食管癌干细胞和贴壁肿瘤细胞辐射增敏效应的差异，并分析食管癌干细胞的辐射抗性及其与相关蛋白表达的关系。方法采用无血清培养基分离细胞系ECA109的食管癌干细胞；流式细胞仪检测细胞表面标记物CD44＋和CD271＋的表达；四甲基偶氮唑盐比色法（MTT）检测NS398联合不同照射剂量（0、4和8Gy）对细胞增殖能力作用的联合效应；克隆形成实验检测NS398对亲本细胞和细胞球辐射增敏效应的差异；Westernblot检测细胞内4种相关蛋白的表达水平。结果在无血清培养基中可分离培养稳定传代的肿瘤干性细胞球。细胞球CD271＋的表达高于亲本细胞（t=3．81，P〈0．05）。照射后细胞球增殖能力大于亲本细胞。NS398联合照射时，亲本细胞的存活分数（SF2）小于细胞球（t=2．91，P〈0．05），NS398对亲本细胞的SER大于细胞球。细胞球内Bmi-1、c．Myc、B-eatenin和CyclinD1的表达水平较亲本细胞均升高（t=8．09、7．90、7．50、7．15，P〈0．05）；4Gy照射后细胞内CyclinDI的表达水平均升高（t=9．74、6．67，P〈0．05）；NS398联合4Gy照射组与4Gy照射组相比，亲本细胞β-catenin和CyclinD1表达水平降低（t=10．15、12．12，P〈0．05），细胞球β-catenin和CyclinD1表达水平亦降低（t=3．23、7．45，P〈0．05）。结论无血清培养基分离培养的食管癌干细胞高表达干细胞表面标记物CD271和干细胞相关蛋白，并具有辐射抗性，其机制可能与β—catenin分子及其下游靶蛋白的表达水平有关。

Objective To study the radiation sensitive enhancement ratio （SER） of NS398 on esophageal cancer stem cells and adherent tumor cells and analyze the radioresistance related protein expressions. Methods ECA109 esophageal cancer stem cells were cultured in serum-free medium. Expression levels of cell surface maker CD44 ＋ and CD271 ＋ were analyzed by flow cytometry. MTT assay was used to detect cell proliferation after the treatments with NS398 and irradiation（0, 4 and 8 Gy）. The sensitization effects of NS398 on the parental cells and its spheroid were evaluated by clone formation assay. Western blot assay was performed to determine protein expressions. Results Serum-free medium was successfully applied to isolate the cancer stem cells with spherical properties. CD271 ＋ in the spheroid cells was notable higher than that in the parent cells （ t = 3.81 , P 〈 0.05 ）. After irradiation, the proliferation rate of parental cells was higher than that in spheroid cells. After the combination treatment of NS398 and irradiation, SF： value of parental cells was lower than spheroid cells （t = 2.91 , P 〈 0. 05 ）and the SER of NS398 on parental cells was greater than spheroid cells. The expressions of Bmi-1 , c-Myc, 6- catenin and Cyclin D1 in spheroid cells were higher than those in parental cells （t = 8.09, 7.90, 7.50, 7. 15,P〈0. 05）. Cyclin D1 expression levels under both cell situations increased after 4 Gy irradiation （t =9.74, 6.67,P 〈0. 05）. Compared to the 4 Gy irradiation alone group,the β-catenin and Cyclin D1 expression levels in both parental cells（ t = 10. 15, 12. 12, P 〈 0.05 ） and spheroid cells （ t = 3.23, 7.45,P 〈 0.05） decreased in the combination group. Conclusions Esophageal cancer stem cells with high level of CD271 can be isolated with serum-free medium and it is radioresistant where β-catenin and its downstream proteins may be involved.