目的 探讨蛇床子素对低分化鼻咽癌CNE2干细胞增殖、放疗敏感性的影响及其机制。方法 采用无血清培养基分离培养低分化鼻咽癌CNE2细胞的干细胞；流式细胞仪检测干细胞表面标记物CD44⁺/CD24^-low的表达和乙醛脱氢酶（ALDH）活性；MTT检测不同质量浓度（0、20、40、80 μg/mL）蛇床子素处理后，CNE2干细胞增殖能力的差异；克隆形成实验检测蛇床子素（40 μg/mL）联合放疗后（0、2、5 Gy），CNE2干细胞克隆形成能力的差异；Western blotting法检测不同质量浓度蛇床子素处理、蛇床子素联合不同放射剂量放疗后，鼻咽癌CNE2干细胞中p-GSK-3β、β-catenin、Cyclin D1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低分化鼻咽癌CNE2干性细胞。细胞CD44⁺/CD24^-low、ALDH⁺的表达高于亲本细胞（P<0.05）；MTT结果显示，与对照组相比，不同质量浓度的蛇床子素作用不同时间后，干细胞增殖抑制率明显增加（P<0.05），且随着蛇床子素质量浓度的增加及作用时间的延长，其抑制作用增强（P<0.05）；克隆形成实验结果显示，与对照组相比，各给药组干细胞克隆形成率均明显降低，蛇床子素+放疗组显著低于蛇床子素组及放疗组（P<0.05）；Western blotting检测结果显示，随着蛇床子素质量浓度的增加，放射剂量的增加，与对照组相比，干细胞p-GSK-3β、β-catenin、Cyclin D1的蛋白表达水平均明显降低，蛇床子素+放疗组显著低于蛇床子素组及放疗组（P<0.05）；结论 蛇床子素能有效抑制低分化鼻咽癌CNE2干细胞的增殖并促进其放疗敏感性，其机制可能是蛇床子素抑制肿瘤干细胞p-GSK-3、β-catenin、Cyclin D1蛋白的表达，从而抑制了干细胞增殖，促进了放疗敏感性。

Objective To investigate the effect and the underlying mechanism of osthole on the proliferation and radiosensitivity of CNE2 stem cells, one of the poorly differentiated cell lines from nasopharyngeal carcinoma. Methods Poorly differentiated CNE2 stem cells were isolated and cultured in serum-free medium (SFM). Flow cytometry was used to detect biomarkers (CD44⁺/CD24^-low) of stem cells and the activity of ALDH. CNE2 stem cells was treated with different concentrations of osthole (0, 20, 40, 80 μg/mL), and then subject to MTT assay. Additionally, CNE2 stem cell was treated with 40 μg/mL osthole plus different dose of radiation (0, 2, 5 Gy) followed by colony formation assay. Consequently, Western blotting was used to detect the difference of pGSK-3β, β-catenin, and Cyclin D1 protein expression in CNE2 stem cells after the treatment of osthole with or without radiation. Results Poorly differentiated CNE2 stem cells isolated and cultured in serum-free medium (SFM) could be passaged stably. The ratio of CD44⁺/CD24^-low and the activity of ALDH were significantly higher in CNE2 stem cells than that in the parental cells (P < 0.05). Compared to the control group, osthole could obviously suppress the proliferation of CNE2 stem cells in a dose and time dependent manner (P < 0.05). Moreover, colony formation assay revealed that inhibition rate of CNE2 stem cell colony formation was highest after the treatment of osthole plus radiation, followed by the treatment of osthole or radiation alone (P < 0.05). Western blotting indicated that in contrast to the controls, the expression of pGSK-3β, β-catenin, and Cyclin D1 protein was mostly down-regulated in the CNE2 stem cells treated with osthole plus radiation, followed by that treated with osthole or radiation alone (P < 0.05). Conclusion Osthole effectively inhibited the proliferation and increased the radiosensitivity of CNE2 stem cells, probably due to the down-regulation of pGSK-3, β-catenin, and Cyclin D1 in tumor stem cells by osthole.

国家自然科学基金青年基金项目（81602886）；广东省自然科学基金项目（2016A030313062）；汕头市科技计划项目（汕府科[2017]166号）