氧浓度及Notch通路对大鼠纤维环细胞增殖和细胞周期的影响

Influences of oxygen tension and Notch signaling pathway on proliferation and cell cycle of rat annulus fibrosus cells

目的检测不同氧浓度条件下椎间盘纤维环细胞Notch信号通路相关分子的表达变化,明确参与调节纤维环细胞增殖的相关靶基因。方法体外分离、培养大鼠纤维环细胞,运用CCK-8细胞活力检测试剂盒、流式细胞仪检测不同氧浓度培养条件下Notch信号通路抑制剂L685458（2μmol/L、4μmol/L、8μmol/L）对纤维环细胞增殖及细胞周期的影响;荧光定量RT-PCR检测不同氧浓度培养条件下纤维环细胞Notch信号表达水平变化。结果 CCK-8结果显示与常氧条件相比,低氧培养纤维环细胞时所测吸光度值明显升高;无论常氧或低氧条件,加入Notch抑制剂干预后所测吸光度值明显降低;流式细胞仪结果显示与常氧条件相比,低氧培养纤维环细胞处于S/G2期细胞所占百分率明显升高,加入Notch抑制剂干预后处于S/G2期细胞所占百分率明显降低。低氧干预8~24 h后,Notch3、Notch4、Delta-like1、Delta-like3、Hes1、Hes5、Hes7 mRNA都有不同水平上调,Hey2 mRNA表达水平下调。结论低氧可以通过上调Notch信号通路的表达水平促进纤维环细胞增殖,Notch信号可能作为一个研究靶点用于延缓椎间盘退变的过程。

Objective To observe the influences of O2 concentration on the expression levels of Notch signaling components in rat annulus fibrosus cells（ AFCs）,and to explore the target gene participates in controlling the proliferation process of AFCs. Methods Rat annulus fibrosus cells were harvested and cultured in vitro under hypoxia or normoxia condition. AFCs were administrated to different concontrations of Notch signal inhibitor,L685458（ 2 μmol / L,4 μmol / L,8 μmol / L）,for 8-24 h,the proliferation and cell cycle of AFCs were examined by CCK-8 assay and flow cytometry. The expression level of Notch signaling components were tested by quantitative real-time polymerase chain reaction（ qRT-PCR）. Results CCK-8 assay indicated that the optical density value of AFCs under hypoxia was increased when compared with under normoxia,while decreased with different concontrations of L685458 whether under hypoxia or normoxia. Flow cytometry indicated that the viability and percentage of S / G2 phase cells of AFCs under hypoxia was increased when compared with under normoxia,while decreased with different concontrations of L685458 whether under hypoxia or normoxia. When AFCs were cultured under hypoxia for 8-24 h,the expression of Notch3,Notch4,Delta-like1,Delta-like3,Hes1,Hes5,Hes7 were increased and Hey2 was decreased at mRNA level compared with under normoxia. Conclusion Hypoxia promotes the proliferation process of AFCs through up-regulation of Notch signaling components,and Notch signaling may become a therapeutic target for retarding the process of disc degeneration.