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目的观察9-顺式维甲酸(9-cis retinoic acid,9-cis RA)联合γ-干扰素(gamma-interfer- on,γ-IFN)在体外诱导神经母细胞瘤(neuroblastoma,NB)SK-N-SH细胞分化、凋亡过程中细胞形态、周期的变化,探讨其协同作用的效果及其机制。方法将细胞分为A(对照组)、B(9-cis RA用药组)、C(γ-IFN用药组)和D(9-cis RA和γ-IFN联合用药组)4组,在处理后适当的时间分别观察细胞形态学变化,进行分化神经节细胞计数,用Hoechst-PI荧光染色法观察细胞的凋亡和死亡,四甲基偶氮唑盐(MTT)法测定NB细胞抑制率,流式细胞仪(flow cytometry,FCM)检测细胞周期分布及其凋亡和死亡。结果联合用药组细胞形态学分化明显,神经节细胞分化率显著高于其他各组(P<0.01);MTT法测定显示联合用药能显著提高NB细胞抑制率并明显优于单独用药;Hoechst-PI荧光染色及FCM钙依赖性磷脂结合蛋白V和碘化丙啶(annexin V/PD染色法检测一致显示联合用药对于诱导NB细胞早期凋亡和坏死具有显著的协同作用;FCM细胞周期动力学分析发现联合用药组G0-G1期细胞比率增加,S期细胞比率减少。结论9-cis RA联合γ-IFN在体外对NB细胞的分化、凋亡和生长抑制能产生明显的协同作用,为其联合应用于临床提供理论依据和指导。
Objective To observe the effects of 9-cis retinoic acid (9-cis RA) combined with gamma-interferon (IFN-γ) on neuroblastoma (NB) N-SH cell differentiation, apoptosis during cell morphology and cycle changes to explore the synergies and its mechanism. Methods The cells were divided into four groups: A (control group), B (9-cis RA treatment group), C (γ-IFN treatment group) and D (9-cis RA and γ-IFN combined treatment group) The cell morphological changes were observed at the appropriate time, the differentiation ganglion cells were counted, the apoptosis and death of cells were observed by Hoechst-PI fluorescent staining, the inhibition rate of NB cells was measured by MTT assay Flow Cytometry (FCM) was used to detect cell cycle distribution and apoptosis and death. Results The morphological differentiation of the combination group was obvious and the differentiation rate of ganglion cells was significantly higher than that of the other groups (P <0.01). The MTT assay showed that the combined treatment could significantly improve the NB cell inhibition rate and was superior to the single drug alone. Hoechst-PI Fluorescent staining and FCM calcium-dependent phospholipid binding protein V and propidium iodide (annexin V / PD staining test showed that the combination of drugs have a significant synergistic effect on induction of early apoptosis and necrosis of NB cells; FCM cell cycle kinetic analysis found The ratio of cells in G0-G1 phase increased and the ratio of S phase cells decreased in combination group.Conclusion The combination of 9-cis RA and γ-IFN has obvious synergistic effect on the differentiation, apoptosis and growth inhibition of NB cells in vitro Provide clinical basis and guidance.