Notch1对神经胶质瘤U251细胞干性及化疗药物敏感性的调节

目的:探讨Notch1对人胶质瘤U251细胞干性和药物敏感性的影响。方法:用高表达Notch1胞内段(Notch1 intracellular domain,NICD1)和Notch1-shRNA慢病毒表达载体感染体外培养的人胶质瘤U251细胞,Western blot和免疫荧光染色法鉴定高表达NICD和Notch1沉默细胞。通过流式细胞术检测分析CD133+细胞的比例、免疫荧光染色法检测nestin和GFAP的表达情况、检测肿瘤细胞球的形成率和SCID小鼠体内种植致瘤情况,分析Notch1对细胞干性的调节。并采用MTT法检测各组细胞对化疗药物替尼泊苷(VM-26)和卡莫司汀(BCNU)的敏感性。结果:NICD表达增加的瘤细胞干性表型增强,如CD133+细胞的比例增加、nestin表达增强而GFAP表达减弱、肿瘤细胞球的形成率和SCID小鼠种植致瘤率增加,并伴有对VM-26和BCNU的敏感性降低。而Notch1基因表达下调的瘤细胞干性表型受到明显抑制,而对VM-26和BCNU的敏感性增高。结论:Notch1高表达可增加人胶质瘤U251细胞的干性,减弱U251细胞对化疗药物的敏感性。

Notch1 regulates stemness and chemotherapeutic sensitivity of human glioma U251 cells

AIM: To investigate whether Notch1 changes stemness and chemotherapeutic sensitivity in human glioma U251 cells. METHODS: The lentiviral vectors, which expressed Notch1-shRNA or Notch1 intracellular domain (NICD), were transfected into U251 cells. Western blot and immunofluorescence staining were applied to monitor the validity of the cells, down-regulation of Notch1 expression or over-expression of NICD. The proportion of CD133⁺ cells was analyzed by flow cytometry. The expression of nestin and GFAP was identified by immunofluorescence staining. The formation rate of tumor cell spheres and the implanted tumor growth in SCID mice were observed. MTT assay was performed to evaluate the chemotherapeutic sensitivity to VM-26 and BCNU of the cells with different treatments. RESULTS: Stemness was significantly enhanced in the cells over-expressing NICD. For example, the proportion of CD133⁺ cells was increased, the expression of nestin was up-regulated, the expression of GFAP was down-regulated, and the formation rate of tumor cell spheres and implanted tumor growth were increased. The chemotherapeutic sensitivity to VM-26 and BCNU of the cells was decreased. In the cells with Notch1 gene down-regulation by RNAi, the stemness was inhibited and chemotherapeutic sensitivity was increased. CONCLUSION: Notch1, which leads to the change of stemness and chemotherapeutic sensitivity in human glioma U251 cells, is likely to be a potential molecular target for treatment of glioma.