mTOR信号通路在神经胶质瘤干细胞样细胞中激活的意义研究

The effect of mTOR pathway on side population cells in glioblastoma

目的分选神经胶质瘤C6细胞中的侧群(SP)细胞,探讨哺乳动物雷帕霉素靶蛋白(mTOR)蛋白对神经胶质瘤SP细胞的影响。方法 Hoechst33342染色流式细胞仪分选SP细胞,另取非侧群(NSP)细胞作为对照。荧光定量-PCR和Western blot分别检测mTOR基因和蛋白的表达。化学合成的双链SiRNA沉默mTOR基因的表达。结果 SP和NSP细胞中mTOR基因的2-△Ct值分别为(0.623±0.087)和(0.289±0.046),两者之间差异有显著性(P<0.01),mTOR蛋白的表达在SP细胞也显著高于NSP细胞。化学合成的靶向SiRNA可以显著下调mTOR的表达(P<0.01)。SiRNA下调mTOR表达后,SP细胞在C6细胞中的比例从5.67±1.46%减少到1.33±0.26%(P<0.01)。结论神经胶质瘤SP细胞高表达mTOR,下调mTOR的表达可以减少SP细胞的比例。

[Objective] To study the effect of mTOR pathway on side population（SP） ceils sorted from glioblas- toma C6 cells. [Methods] Hoechst33342 staining and flow cytometric sorter were used to sort SP and NSP cells. The mRNA and protein expression of mTOR were determined by Real-time PCR and Western blot analysis, respec- tively. The small interfering RNA was used to specifically knockdown mTOR in glioblastoma C6 cells. [Results] The 2-ACt values of mTOR was （0.623±0.087） in SP cells by Real-time PCR analysis, which was higher than （0.289±0.046） in NSP cells （P 〈0.01）. The protein expression of roTOR in SP cells was also significantly higher than that in NSP ceils. The mRNA and protein expression of roTOR was significantly and specifically depleted by SiRNA duplexes （P 〈0.01）. After treatment with roTOR specific SiRNA duplexes, the percentage of SP cells in glioblastoma C6 cells was decreased from （5.67±1.46）% to （1.33±0.26）%（P 〈0.01）. [Conclusion] The mTOR pathway plays an important role in the SP cells phenotype of glioblastoma. The downregulation of mTOR decreased the percentage of SP cells in glioblastoma cells.