CAJ | 학술논문

目的探索奥沙利铂对不同p53状态肝癌细胞的DNA损伤修复基因(GADD45β)诱导差异及可能调控机制.方法转染p53全序列建立Hep3B+p53.体外合成GADD45β近端启动子序列群(-618至-314),构建荧光素表达质粒,转染肝癌细胞株HepG2、Hep3B和Hep3B+p53.以实时荧光定量PCR比较奥沙利铂对GADD45β表达诱导及其近端启动子活性影响差异；比较对DNA合成和细胞克隆形成能力抑制差异；通过Caspase-3的表达变化测定凋亡的发生和发展.结果奥沙利铂对Hep3B中GADD45β诱导并不明显.转染p53全基因序列后,Hep3B+p53对奥沙利铂的敏感性明显增加,并呈现出剂量-效应的正相关关系.荧光素分析提示奥沙利铂作用Hep3B+p53后的启动子NF-κB(-618/+6)和E2F-1(-470/+6)活性较Hep3B明显增强约1.5倍和0.8倍.奥沙利铂能够更加明显地抑制Hep3B+p53的DNA合成能力和细胞克隆形成能力,100 μmol/L奥沙利铂作用后Hep3B+p53DNA合成率为30.41％,对细胞克隆形成能力的抑制率则达到75.60％,与Hep3B相比差异显著.奥沙利铂作用后Hep3B+p53的Caspase-3能够迅速地启动凋亡的发生和发展.结论 p53对铂类化疗药物对肝癌细胞的GADD45β诱导具有重要作用,增强转录调节因子的表达水平是其可能的作用机制.
목적 탐색오사리박대불동p53상태간암세포적DNA손상수복기인(GADD45β)유도차이급가능조공궤제.방법 전염p53전서렬건립Hep3B+p53.체외합성GADD45β근단계동자서렬군(-618지-314),구건형광소표체질립,전염간암세포주HepG2、Hep3B화Hep3B+p53.이실시형광정량PCR비교오사리박대GADD45β표체유도급기근단계동자활성영향차이；비교대DNA합성화세포극륭형성능력억제차이；통과Caspase-3적표체변화측정조망적발생화발전.결과 오사리박대Hep3B중GADD45β유도병불명현.전염p53전기인서렬후,Hep3B+p53대오사리박적민감성명현증가,병정현출제량-효응적정상관관계.형광소분석제시오사리박작용Hep3B+p53후적계동자NF-κB(-618/+6)화E2F-1(-470/+6)활성교Hep3B명현증강약1.5배화0.8배.오사리박능구경가명현지억제Hep3B+p53적DNA합성능력화세포극륭형성능력,100 μmol/L오사리박작용후Hep3B+p53DNA합성솔위30.41％,대세포극륭형성능력적억제솔칙체도75.60％,여Hep3B상비차이현저.오사리박작용후Hep3B+p53적Caspase-3능구신속지계동조망적발생화발전.결론 p53대박류화료약물대간암세포적GADD45β유도구유중요작용,증강전록조절인자적표체수평시기가능적작용궤제.
Objective To identify the role of p53 in the induction of growth arrest DNA damage-inducible gene 45β (GADD45β) in HCC cells by Oxaliplatin.Methods A Hep3B+p53 clone was established by transfection of the full-length p53 sequence to Hep3B.Following oxaliplatin administration,quantitative real-time PCR was employed to validate the expression changes of GADD45β.pGL3 basic luciferase plasmids including promoter fragments were synthesized in vitro and transfected into cells.The effects on promoter activity,cell growth and the cleavage of Caspase-3 were further focused on.Results Hep3B+p53 expressed p53 protein stably.The transfection of p553 enhanced the induction of GADD45β in Hep3B by Oxaliplatin.The promoter activity of fragments constructed NF-κB and E2F-1 binding sites was induced about 1.5 and 0.8 folds by transfection of p53.The colony formation and DNA syntheses were inhibited apparently in Hep3B+p53 with p53 by Oxaliplatin (30.41％ and 75.60％ by 100 μmol/L Oxaliplatin,respectively).Moreover,p53 transfection triggered cleavage of Caspase-3 more rapidly.Conclusion p53 played a role in the induction of GADD45β in Hep3B by Oxaliplatin.