封闭Notch信号影响神经干细胞分化的体外研究

DAPT inhibits transforming growth factor-β1-induced differentiation of neural stem cells via blocking Notch signaling

目的探讨γ-分泌酶抑制剂N-[N-(3,5-二氟苯乙酰丙烯基)]-s-苯甘氨酸叔丁基酯(DAPT)对转化生长因子-β1(TGF-β1)诱导神经干细胞(NSCs)分化的作用及对Notch通路的影响。方法原代培养来自大鼠前脑组织的NSCs,根据处理措施将NSCs分为溶剂对照组、TGF-β1诱导组(TGF-β1处理细胞,浓度为5 ng/L)和DAPT干预组(5 ng/L TGF-β1基础上加入1或10μmol/L DAPT)。细胞培养3、8、24和48 h后,real-time RT-PCR检测Notch信号关键分子Notch1和Jagged1 mRNA的表达;WesternBlot检测星形胶质细胞标志物-胶质纤维酸性蛋白(GFAP)及NSCs标志物-巢蛋白(Nestin)的表达;细胞免疫荧光染色法检测Notch1、Jagged1、GFAP及Nestin的表达。结果TGF-β1处理后,可加速诱导NSCs的分化。NSCs标记物GFAP的表达呈现时间依赖性,早期并不表达,在8 h后GFAP开始表达,并随时间延长而表达增加。而Nestin早期明显表达,但8 h后表达明显下调,这提示(TGF-β1)作用8 h后可诱导NSCs向星形胶质细胞转化。深入研究显示,Notch1和Jagged1也在TGF-β1作用8 h后表达逐渐升高,这提示TGF-β1介导星形胶质细胞转化过程中,Notch信号可能被激活进而参与了此过程。应用DAPT处理后,GFAP表达在8 h与对照组相比无明显差异,而在48 h后逐渐下降;伴随着Notch1和Jagged1表达的下调,提示DAPT作用后,NSCs向星形胶质细胞转化被抑制,这与Notch信号活性的下降有关。结论TGF-β1可诱导了NSCs向星形胶质细胞转化,并且具有时间依赖性;其机制可能与Notch信号的活化有关。DAPT的干预可封闭Notch信号,进而抑制TGF-β1介导NSCs向星形胶质细胞分化。

【Abstract】Objective To investigate the effect of theγ-secretase inhibitor N[N(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester(DAPT)on the transforming growth factorβ1(TGFβ1)-induced differentiation of neural stem cells(NSCs)and Notch signaling.Methods Primary cultured NSCs were obtained from rat forebrain tissue.NSCs were divided stochastically into a control group(treated with vehicle only),a TGF-β1 group(treated with TGF-β1 at 5 ng/L),and a DAPT group(treated with TGF-β1 at 5 ng/L plus DAPT at 1 or 10μmol/L).After culture for 3,8,24,and 48 h,the mRNA expression of Notch1 and Jagged1 was quantified by real-time RT-PCR.The protein expression of the astrocyte marker glial fibrillary acidic protein(GFAP)and the NSC marker Nestin was determined by Western blot.The expression and location of Notch1,Jagged1,GFAP,and Nestin in NSCs were detected by immunofluorescence staining.Results After TGFβ1 treatment,the differentiation of NSCs was accelerated.At the early stage,Nestin was not expressed in NSCs.After 8 h,GFAP began to be expressed and increased with time.Conversely,Nestin was expressed in NSCs at the early stage,but it was down-regulated after 8 h,suggesting that TGF-β1 can induce the transformation of NSCs into astrocytes after 8 h.Further studies showed that the expression of Notch1 and Jagged1 was increased gradually after TGF-β1 treatment for 8 h,suggesting that TGF-β1 mediated the activation of Notch signaling during astrocyte transformation.After treatment with DAPT,GFAP expression was not inhibited at 8 h,but decreased gradually after 48 h,accompanied by down-regulation of Notch1 and Jagged1,suggesting that the transformation of NSCs to astrocytes was inhibited after DAPT treatment.The mechanism may be associated with the decrease in Notch signaling activity.Conclusion TGF-β1 induces the transformation of NSCs into astrocytes in a time-dependent manner;its mechanism may be related to the activation of Notch signaling.Intervention with DAPT blocks Notch signaling,which inhibits TGFβ1-mediated differentiation of NSCs into astrocytes at a certain time point.