中文摘要：

目的 探讨3-氨基吡啶-2-甲醛硫代缩氨基脲（Triapine）对不同p53状态肝癌细胞的生长抑制及DNA损伤诱导基因45β（GADD45β）诱导差异及其调控机制.方法 转染p53全序列建立Hep3B＋p53；体外合成GADD45β近端6个启动子序列群（-618 ～-314）,构建荧光素表达质粒,转染肝癌细胞株HepG2、Hep3B和Hep3B＋p53；以实时荧光定量聚合酶链反应（FQ-PCR）比较Triapine对GADD45β表达诱导及其近端启动子活性影响差异；进一步比较对DNA合成和细胞克隆形成能力抑制差异；并通过半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3的表达变化测定细胞凋亡的发生和发展.结果 Triapine对Hep3B中GADD45诱导并不明显,Hep3B＋p53对Triapine敏感性明显增加,2.5、5.0μmol/L剂量下GADD45/甘油醛-3-磷酸脱氢酶（GAPDH）比值分别为0.0425和0.0704,并呈现出剂量-效应的正相关关系.荧光素分析提示Triapine作用Hep3B＋p53后的启动子核因子（NF）-κB（-618/＋6）和E2F-1（-470/＋6）活性较Hep3B明显增强约1.3倍和0.7倍.Triapine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,5μmol/L Triapine对Hep3B ＋p53DNA合成的抑制率为40.89％,对细胞克隆形成能力的抑制率达到68.16％,与Hep3B比较差异有统计学意义（P＜0.05）.Triapine作用后Hep3B＋p53的Caspase-3能迅速激活,活性高峰提前出现于6h.结论 p53在核糖核苷酸还原酶抑制剂化疗药物对肝癌细胞的GADD45诱导中具有重要作用,其作用机制可能是增强转录调节因子的表达水平.

英文摘要：

Objective To identify the role of p53 in the induction of growth arrest DNA damageinducible gene 45 β （GADD45β） in hepatocellular carcinoma （HCC） cells by Triapine.Methods Hep3B ＋p53 clone was established by the transfection of the full-length p53 sequence to Hep3B.Following Triapine administration,real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） were employed to validate the expression changes of GADD45β.pGL3 basic luciferase plasmids including six promoter fragments （-618/-314） were synthesized in vitro and transfected into cells.The effects on promoter activity,cell growth and the cleavage of cysteinyl aspartate-specific protease （Caspase）-3 were further focused on.Results GADD45β could be induced apparently in Hep3B＋p53 by Triapine,which couldn＇t be enhanced in Hep3B.GADD45/glyceraldehyde-3-phosphate dehydrogenase （GAPDH） increased to 0.0425 and 0.0704 by 2.5 and 5.0 μmol/L Triapine,respectively.The promoter activity of nuclear factor-κB （NF-KB） and E2F-1 binding sites could be induced about 1.3 and 0.7 folds by transfection of p53.The colony formation and DNA syntheses were inhibited apparently in Hep3B＋p53 by Triapine （40.89％ and 68.16％ by 5 μmol/L Triapine,respectively）.Moreover,the p53 transfection could trigger the cleavage of Caspase-3 more rapidly.Conclusion p53 may play an important role in the induction of GADD45β in Hep3B by Triapine.