桑辛素对喉癌干细胞干性表型调控的作用研究

The Effect of Morusin on Stemness Phenotype of Laryngeal Cancer Stem Cell

目的探索桑辛素对喉癌干细胞干性表型调控的影响。方法流式细胞仪分选并检测CD133^+喉癌干细胞比例;肿瘤球形成实验检测CD133^+喉癌干细胞的自我更新能力;Transwell实验检测CD133^+喉癌干细胞的迁移能力;改良MTT实验检测化疗药物对CD133^+喉癌干细胞的细胞毒性作用;免疫荧光染色、实时荧光定量PCR(RT-qPCR)及Western blot检测CD133^+喉癌干细胞的干细胞标志物表达;在不同浓度的桑辛素处理CD133^+喉癌干细胞后(以桑辛素0μmol/L为对照),通过肿瘤球形成实验、Transwell实验、改良MTT实验及Western blot,分别检测CD133^+喉癌干细胞的自我更新能力、迁移能力、化疗药物的细胞毒性作用及干细胞标志物表达变化。结果流式细胞仪分选结果显示,CD133^+喉癌干细胞占喉癌细胞的比例为(3.50±0.34)%;经过培养富集后,其比例可达(93.20±5.23)%。肿瘤球形成实验结果显示,与喉癌细胞相比,CD133^+喉癌干细胞具有增强的自我更新能力(P<0.001);Transwell实验显示,与喉癌细胞相比,CD133^+喉癌干细胞的迁移能力增强(P<0.05);改良MTT实验结果显示,与喉癌细胞相比,CD133^+喉癌干细胞抵抗化疗药物(5-氟尿嘧啶及顺铂)的细胞毒性作用(P<0.05);免疫荧光染色、RT-qPCR及Western blot结果显示,干细胞标志物(CD133、ALDH1、Sox2、ABCG2及N-cadherin)在CD133^+喉癌干细胞中呈高表达水平。通过不同浓度的桑辛素处理CD133^+喉癌干细胞,其自我更新能力降低(P<0.05);迁移能力亦下降(P<0.05);此外,桑辛素处理的CD133^+喉癌干细胞对化疗药物的细胞毒性作用更加敏感(P<0.05);Western blot结果显示,不同浓度的桑辛素处理的CD133^+喉癌干细胞,其上述的干细胞标志物表达水平下调(P<0.05)。结论 CD133^+喉癌干细胞具有干性表型特征;桑辛素可减弱喉癌干细胞的干性表型,可能与下调其干细胞标志物表达相关。

Objective To investigate the regulation effect of Morusin on stemness phenotype of laryngeal cancer stem cells. Methods Separation and detection the proportion of CD133^+ laryngeal cancer stem cells through flow cytometry;evaluation the self-renewal ability of CD133^+ laryngeal cancer stem cells by tumor sphere formation assay;exploring the migration ability of CD133^+ laryngeal cancer stem cells by Transwell assay;analyzing the cytotoxicity of chemotherapy drugs on CD133^+ laryngeal cancer stem cells by modified MTT assay;detection of the expression levels of stemness associated markers by immunofluorescence staining, RT-qPCR and Western blot. After treatment with different concentrations of Morusin, cells were performed the above experiments for detection the self-renewal ability, migration ability, cytotoxicity resistance and expression of stemness associated markers. Results Flow cytometry analysis showed that the proportion of CD133^+ laryngeal cancer stem cells was(3.50±0.34)%, while after enrichment, the proportion increased to(93.20±5.23)%. CD133^+ laryngeal cancer stem cells exhibited better self-renewal ability(P<0.001) and migratory ability(P<0.05);they were resistant to the cytotoxicity of chemotherapy drug(P<0.05), and highly expressed of stemness associated markers. After being treated with Morusin, the self-renewal and migratory abilities of CD133^+laryngeal cancer stem cells were reduced(P<0.05). In addition, after treated with Morusin, CD133^+ laryngeal cancer stem cells were more sensitive to chemotherapy drugs;moreover, the expression levels of stemness associated markers were decreased. Conclusion CD133^+ laryngeal cancer stem cells possessed stemness phenotypic characteristics. Morusin attenuated stemness phenotype of laryngeal cancer stem cells, which may be related to its down-regulation effect on stemness associated markers.