Celecoxib联合羟基喜树碱对缺氧诱导的人肝癌细胞增殖与凋亡的影响

目的:探讨选择性环氧合酶-2(COX-2)抑制剂Celecoxib联合羟基喜树碱对缺氧诱导的人肝癌细胞株SMMC-7721细胞增殖和凋亡的影响。方法:应用三气培养箱在缺氧环境下培养人肝癌细胞株SMMC-7721细胞。预实验筛选Celecoxib、羟基喜树碱最小有效终浓度，设24、48、72小时为干预时间并筛选最佳作用时间。分别以Celecoxib及羟基喜树碱单独实验筛选的最小有效浓度为联合组浓度；以Celecoxib单独实验筛选最佳作用时间为实验时间。倒置显微镜观察细胞形态学变化；应用四氮唑盐比色法(MTT法)监测细胞增殖活力；Annexin V/PI双染色法检测细胞凋亡；免疫细胞化学法（SP法）检测凋亡蛋白Bax、Bcl-2、COX-2的表达。结果:不同浓度Celecoxib及不同时间对SMMC-7721细胞表现出的生长抑制作用呈剂量依赖性及时间依赖性。Celecoxib（30 mg/L)单用在作用72 h时即可抑制SMMC-7721细胞的增殖。选72 h为作用时间，不同浓度羟基喜树碱对SMMC-7721细胞表现出的生长抑制作用呈剂量依赖性，浓度为0.625 μmol/L羟基喜树碱即可抑制SMMC-7721细胞的增殖。选用羟基喜树碱(0.625 μmol/L)与Celecoxib（30 mg/L)联合作用72 h时，联合抑制作用结果呈现协同作用(Q>1.15)。流式细胞仪检测分析，两种药物均可有效诱导 SMMC-7721细胞的凋亡，并且在上述浓度联合作用时具有协同效应。免疫细胞化学结果显示，Celecoxib（30 mg/L)组和联合用药组细胞质内棕黄色颗粒较对照组染色浅，表明Bcl-2和COX-2蛋白表达有所下降，而Bax蛋白表达在Celecoxib（30 mg/L)和联合用药组中黄色颗粒的阳性细胞较对照组增多，表明Bax蛋白表达有所上调；并且此变化与Celecoxib（30 mg/L)组比较联合用药组有协同作用(P<0.05)。结论:缺氧状态下Celecoxib与羟基喜树碱对SMMC-7721细胞株均有抑制增殖与促进凋亡作用，并且两者联合作用有协同作用；Celecoxib的作用机制除了抑制COX-2外，还可能与下调Bcl-2、上调Bax的表达有关。

The effects of Celecoxib combined with Hydroxycamptothecine on proliferation and apoptosis of human hepatocarcinoma cells induced by hypoxia

Objective:To investigate the effects of selective cyclooxygenase-2（COX-2) inhibitor Celecoxib combined with Hydroxycamptothecine on proliferation and apoptosis of human hepatocarcinoma cell line SMMC-7721 induced by hypoxia.Methods:Human hepatocarcinoma cell line SMMC-7721 was cultured in a three-gas incubator to establish a hypoxic cell model.The minimum effective final concentrations of Celecoxib and Hydroxycamptothecine were decided by pre-experiment，and the optimal action time was selected for the intervention time at 24，48，and 72 hours.The combined group used the minimum effective concentration of Celecoxib alone and the minimum effective concentration of Hydroxycamptothecine alone.The best time was selected by Celecoxib experiment alone.The morphology of the cells was observed by inverted microscope.The cell proliferation was observed by MTT assay.The cell apoptosis was detected by Annexin V/PI double staining.The apoptosis protein expression of Bax，Bcl-2，COX-2 were detected by immunocytochemistry（SP method).Results:Different concentrations and time of Celecoxib showed that the growth inhibition of SMMC-7721 cells were dose-dependent and time-dependent.Celecoxib（30 mg/L) alone could inhibit the proliferation of SMMC-7721 cells at 72 h.So 72 h was selected as the action time.The inhibitory effect of different concentrations of Hydroxycamptothecine on SMMC-7721 cells was dose-dependent.The concentration of 0.625 μmol/L Hydroxycamptothecine could inhibit the proliferation of SMMC-7721 cells.When the combination of Hydroxycamptothecine（0.625 μmol/L) and Celecoxib（30 mg/L) for 72 h，the combined inhibition showed a synergistic effect（Q>1.15).Flow cytometry showed that both drugs could effectively induce SMMC-7721 cells apoptosis，and synergistic effect when combined with the two drugs.Immunocytochemistry results showed that the cytoplasmic brown-yellow in the cells of the Celecoxib（30 mg/L) group and the combination group was lightly stained，indicating that the expression of Bcl-2 and COX-2 protein was decreased，while Bax protein expression in the Celecoxib（30 mg/L) group and the combination group increased compared with the control group，indicating that the Bax protein expression was up-regulated.And this change had synergistic effect when the combination group compared with the Celecoxib（30 mg/L) group（P<0.05).Conclusion:Celecoxib and Hydroxycamptothecine can inhibit proliferation and promote apoptosis of human hepatocarcinoma cell line SMMC-7721 under hypoxia，and combination of the two drugs can have the synergistic effect.Besides the inhibition of COX-2，the inhibition effect of Celecoxib maybe associated with the decreased expression of Bcl-2 and increased expression of Bax.