Notch1对神经胶质瘤U251细胞干性及化疗药物敏感性的调节

Notch1 regulates stemness and chemotherapeutic sensitivity of human glioma U251 cells

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。

AIM： To investigate whether Notchl changes stemness and chemotherapeutic sensitivity in human glioma U251 cells. METHODS： The lentiviral vectors, which expressed Notchl-shRNA or Notchl intracellular domain （NICD）, were transfected into U251 cells . Western blot and immunofluorescence staining were applied to monitor the va- lidity of the cells, down-regulation of Notchl 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 forma- tion rate of tumor cell spheres and the implanted tumor growth in SCID mice were observed. MTF assay was performed to e- valuate the chemotherapeutic sensitivity to VM-26 and BCNU of the ceils with different treatments. RESULTS ： Sternness 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 ceils with Notchl gene down-regulation by RNAi, the stemness was inhibited and chemotherapeutic sensi- tivity was increased. CONCLUSION： Notchl, 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.