摘要目的 探讨特异性环氧化酶-2(COX-2)抑制剂塞来昔布(celecoxib)对人神经母细胞瘤细胞系SH-SY-5Y细胞生长周期的影响及其分子生物学作用机制.方法 不同浓度组塞来昔布处理SH-SY-5Y细胞,倒置显微镜观察药物作用后的细胞形态学变化,MTT法检测各浓度组作用48 h后细胞的增殖情况,流式细胞仪检测不同浓度塞来昔布作用后对细胞周期的影响,Western blot检测药物作用前后细胞周期蛋白CyclinD1、P16、P21蛋白表达的变化.结果 ①不同浓度塞来昔布作用后,倒置显微镜观察到细胞形态学改变,SH-SY-5Y细胞为细长的梭形,少数呈类三角形,随着药物浓度的增加,细胞变短、变圆.整个细胞的形态由梭形逐渐变为类多角形,最后细胞间隙增大,细胞变圆;②MTT法显示,塞来昔布以10、20、40、80μmol/L分别作用于SH-SY-5Y细胞48 h时,其细胞增殖率分别为(90.1±3.6)%、(69.3±3.7)%、(44.2±4.6)%、(10.2±2.9)%,不同浓度组间比较,差异有统计学意义(P＜0.05),塞来昔布在一定剂量范围内以剂量依赖性的方式抑制SH-SY-5Y细胞的生长;③流式细胞仪检测发现随药物作用浓度增加,当塞来昔布以0、10、20、40、80 ttmol/L分别作用于SH-SY-5Y细胞48 h时,G0/G1期细胞比例分别为(50.7±4.0)%,(55.1±5.2)%,(67.2±3.9)%,(71.1±3.8)%,(75.5±4.8)%,其中20、40、80μmol/L组与未加药组比较,差异有统计学意义(P＜0.01);④Western blot显示随着药物浓度的增加,细胞中CyclinD1蛋白的表达水平逐渐降低,细胞巾P21、P16蛋白的表达水平逐渐增加.结论 塞来昔布抑制SH-SY-5Y细胞的牛长并阻滞细胞牛长在G1期,其机制与通过抑制细胞周期蛋白CyclinD1的表达及增加细胞周期蛋白抑制因子P21、P16表达可能有关.塞来昔布可望成为神经母细胞瘤治疗的新的药物.

abstractsObjective To investigate the influence of high selective Cyclooxygenase-2 (COX-2) inhibitor celecoxib on proliferation and cell cycle of SH-SY-5Y cells and explore the potential COX-2dependent mechanism of celecoxib on the influence of SH-SY-5Y.Methods The SH-SY-5Y cells were treated by celecoxib with different doses(10 μmol/L,20 μmol/L,40μmol/L,80 μmol/L) for 48 hours.Cellular morphology was observed via inverted microscope.MethaberLzthiazuron (MTT) assay was used to evaluate proliferation of cells.The effect of celecoxib with different doses on cell cycle was detected by flow cytometry (10μmol/L,20 μmol/L,40 μmol/L,80 μmol/L).Western blot analysis for expression of CyclinD1,P16 and P21 was performed before and inoculation of celecoxib with different doses(10μmol/L,20μmol/L,40μmol/L,80μmol/L).Results After SH-SY-SY cells were treated by celecoxib with different doses (10 μmol/L,20 μmol/L,40 μmol/L,80 μmol/L) for 48 hours,the shape of SH-SY-5Y cells changed significantly.The shape of SH-SY-5Y cells pre-treated with celecoxib was in slender and fusiform shape.SH-SY-5Y cells became short or polygon after the treatment by celecoxib with concentrations.MTT assay showed the proliferation rate of SH-SY-5Y was 90.1%±3.6%,69.3% + 3.7% ,44.2% + 4.6% and 10.2% + 2.9% respectively with the dose of 10 μmol/L,20 μmol/L,40μmol/L and 80 μmol/L.Celecoxib inhibited the cells' proliferation in dose dependence.Flow cytometry showed the ratio of G0/G1 was 50.70% ± 4.0%,55.10%±5.2 %,67.20 %±3.9%,71.07 %±3.8% and 75.53%±4.8% respectively with the dose of 0μmol/L,10μmol/L,20 μmol/L,40 μmol/L and 80 μmol/L,in which the dose of 20 μmol/L,40 μmol/L and 80 μmol/L could significantly increase the ratio of G0/G1 compared with controls(P＜0.01).The expression of CyclinDl,P21 and P16 were detected in SH-SY-SY cells.The western blot showed the expression level of CyclinD1 decreased with the increasing of the dose of celecoxib,while P21 and PI6 increased.Conclusions Celecoxib inhibits growth of SH-SY-5Y cells in G1 by down-regulating the expression of CyclinD1 and up-regulating the expression of P16 and P21 in vitro.Celecoxib would become a promising drug as an adjuvant therapy for children with neuroblastoma.