双氯芬酸的抗增殖作用与细胞内环氧酶-2表达水平的关系

Association of cyclooxygenase-2 expression and antiproliferation effect of diclofenac in different cells

目的检测体外培养的人肝细胞癌HepG2、Hep3B细胞和正常肝细胞系L02、QSG7701细胞以及正常人皮肤成纤维细胞Fb环氧酶-2（cyclooxygenase-2，COX-2）mRNA的表达，观察环氧酶抑制剂双氯芬酸（diclofenac）对上述细胞增殖的影响，以探讨双氯芬酸抗增殖作用与细胞内COX-2表达的关系。方法采用半定量逆转录聚合酶链反应（RT—PCR）检测各种细胞系COX-2mRNA的表达水平，采用cellcounting kit-8（CCK-8）细胞计数法测定药物作用72h后的细胞增殖活性，绘制生长曲线。结果肝细胞癌HepG2、Hep3B细胞和正常肝细胞L02均高表达COX-2mRNA，正常肝细胞QSG7701微弱表达COX-2mRNA，正常人皮肤成纤维细胞Fb不表达COX-2mRNA。双氯芬酸在10～200μmol·L^-1内呈浓度依赖性地抑制HepG2、Hep3B和L02细胞的增殖，作用72h的半数抑制浓度分别为76．41、35．21和87．44μmol·L^-1。双氯芬酸对QSG7701的抑制作用较弱，半数抑制浓度为170．23μmol-L^-1，对Fb细胞仅在浓度超过200μmol·L^-1时才表现出细胞毒性。结论环氧酶抑制剂双氯芬酸特异性地抑制COX-2mRNA高表达的肝细胞癌HepG2、Hep3B细胞和正常肝细胞L02的增殖．提示双氯芬酸通过COX-2途径起抗增殖作用。

Objective To investigate the cyclooxygenase-2（COX-2） mRNA expression of cultured hepatocellular carcinoma call lines HepG2, Hep3B, human hepatocellular call line QSG 7701, L02 and human skin fibroblast Fb, and detect the effects of the cyclooxygenase inhibitor diclofenac on proliferation of those cells. Methods COX-2 mRNA expression was determined by semi-quantitative reverse transcription polymerase chain reaction（RT-PCR）, and cell proliferation analyzed by cell counting kit-8（CCK-8） assay after exposure to diclofenac at various concentrations （10 - 200μmol. L^-1） for 72 h. Results COX-2 mRNA expression was observed in HepG2, Hep3B and L02 ceils, but hardly in QSG7701 and not in Fb cell. 72h of didofenac exposure significandy inhibited the HepG2, Hep3B and L02 cells proliferation in a concentration-dependent manner. The 50 percent inhibitory concentration （IC50） of diclofenac was 76.41,35.21 and 87.44 μmol.L^-1 respectivdy. A much weaker antiproliferation effect on QSG7701 was shown, with IC50 being 170.23 μmol. L^-1. Diclofenac could not suppressed Fb cell growth until its concentration reached at 200μmol. L^- 1. Conclusions Diclofenac can specially inhibit the proliferation of COX-2 expressing cells HepG2 , Hep3B and L02, which indicates the association of COX-2 expression and the antiproliferative effect of diclofenac.