Notch信号通路在1-甲基-4-苯基吡啶诱导的人神经母细胞瘤细胞凋亡中的作用

Notch signaling pathway in 1-methyl-4-phenylpyridinium-induced apoptosis of SH-SY5Y cell

目的 探讨Notch信号通路在1-甲基-4-苯基吡啶（MPP＋）诱导的入神经母细胞瘤细胞凋亡中的活性变化及Notch信号通路抑制剂DAPT对凋亡的调控作用.方法 （1）体外培养的SHSY5Y与不同浓度（0、0.5、1、1.5、2 mmol/L）的MPP＋共同孵育0、24、48、72 h.Annexin V-FITC/PI双染及流式细胞术检测细胞凋亡,Western印迹法检测Notch信号通路相关蛋白Notch-1、Jagged-1及Hes-1的表达.（2）将10 μmol/L的γ-分泌酶抑制剂（DAPT）预处理15 min后的SH-SY5Y细胞与1.5 mmol/LMPP＋共同孵育48 h,Annexin V-FITC/PI双染及流式细胞术检测细胞凋亡,Western印迹法检测Notch-1、Jagged-1、Hes-1蛋白表达的变化.结果 MPP＋以剂量（3.20％±0.19％、10.00％±1.72％、20.60％±3.76％、32.80％±5.12％、46.00％±5.06％,均P＜ 0.05）和时间（2.80％±0.21％、12.30％±1.82％、19.60％±2.89％、35.00％±4.78％,均P＜ 0.05）依赖方式诱导SH-SY5Y细胞凋亡,并显著上调Notch信号通路相关蛋白Notch-1、Jagged-1、Hes-1的表达.DAPT可显著抑制MPP＋诱导的SH-SY5Y细胞凋亡（3.10％ ±0.21％比35.50％±4.98％、19.20％±2.98％,均P＜ 0.05）以及Notch-1、Jagged-1、Hes-1蛋白的表达.结论 MPP＋诱导SH-SY5Y细胞凋亡中Notch信号通路活化,DAPT可阻断Notch信号通路激活,减少MPP＋诱导的SH-SY5Y细胞凋亡.

Objective To explore the role of Notch signaling pathway and the effect of γ-secretase inhibitor （DAPT） on the apoptosis induced by 1-methyl-4-phenylpyridinium （MPP＋） in differentiated SHSY5Y cell.Methods SH-SY5Y cell were incubated with various concentrations （0,0.5,1,1.5,2 mmol/L） of MPP＋ for 0,24,48 and 72 h respectively.Flow cytometry with Annexin V-FITC/PI double staining was used for apoptotic analysis.The protein expressions of Notch-1,Jagged-1 and Hes-1 were detected by Western blot.SH-SY5Y cell were preincubated with 10 μmol/L DAPT for 15 min before 1.5 mmol/L MPP＋ treatment for 48 h.Flow cytometry and Western blot were performed to analyze the cell apoptosis and protein expressions of Notch-1,Jagged-1 and Hes-1.Results MPP＋ induced the apoptosis of SH-SY5Y cell in a dose （3.20％ ±0.19％ vs 10.00％ ± 1.72％,20.60％ ±3.76％,32.80％ ±5.12％,46.00％ ±5.06％,all P＜0.05） and time-（2.80％ ±0.21％ vs 12.30％ ± 1.82％,19.60％ ±2.89％,35.00％ ± 4.78％,all P ＜ 0.05） dependent manner.MPP ＋ up-regulated the expressions of Notch-1,Jagged-1 and Hes-1 in a dose-and time-dependent manner in SH-SY5Y cell.DAPT treatment decreased MPP＋-induced apoptosis （3.10％ ± 0.21％ vs 35.50％ ± 4.98％,19.20％ ± 2.98％,both P ＜ 0.05） and the expressions of Notch-1,Jagged-1 and Hes-1 in SH-SY5Y cell.Conclusion The activation of Notch signaling pathway plays an important role in MPP＋-induced apoptosis of SH-SY5Y cell.DAPT inhibits Notch signaling pathway and protects SH-SY5Y cell from MPP＋-induced apoptosis.