中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
10期
2029-2031
,共3页
葛覃%房嫣%杨卫平%付志平%李科%佟辉%施敏敏%邱伟华
葛覃%房嫣%楊衛平%付誌平%李科%佟輝%施敏敏%邱偉華
갈담%방언%양위평%부지평%리과%동휘%시민민%구위화
肝癌%3-氨基吡啶-2-甲醛硫代缩氨基脲%生长抑制及DNA损伤诱导基因45β%p53%启动子
肝癌%3-氨基吡啶-2-甲醛硫代縮氨基脲%生長抑製及DNA損傷誘導基因45β%p53%啟動子
간암%3-안기필정-2-갑철류대축안기뇨%생장억제급DNA손상유도기인45β%p53%계동자
Hepatocellular carcinoma%Triapine%Growth arrest DNA damage-inducible gene 45β%p53%Promoter
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.
目的 探討3-氨基吡啶-2-甲醛硫代縮氨基脲(Triapine)對不同p53狀態肝癌細胞的生長抑製及DNA損傷誘導基因45β(GADD45β)誘導差異及其調控機製.方法 轉染p53全序列建立Hep3B+p53;體外閤成GADD45β近耑6箇啟動子序列群(-618 ~-314),構建熒光素錶達質粒,轉染肝癌細胞株HepG2、Hep3B和Hep3B+p53;以實時熒光定量聚閤酶鏈反應(FQ-PCR)比較Triapine對GADD45β錶達誘導及其近耑啟動子活性影響差異;進一步比較對DNA閤成和細胞剋隆形成能力抑製差異;併通過半胱氨酰天鼕氨痠特異性蛋白酶(Caspase)-3的錶達變化測定細胞凋亡的髮生和髮展.結果 Triapine對Hep3B中GADD45誘導併不明顯,Hep3B+p53對Triapine敏感性明顯增加,2.5、5.0μmol/L劑量下GADD45/甘油醛-3-燐痠脫氫酶(GAPDH)比值分彆為0.0425和0.0704,併呈現齣劑量-效應的正相關關繫.熒光素分析提示Triapine作用Hep3B+p53後的啟動子覈因子(NF)-κB(-618/+6)和E2F-1(-470/+6)活性較Hep3B明顯增彊約1.3倍和0.7倍.Triapine能夠更加明顯地抑製Hep3B +p53的DNA閤成能力和細胞剋隆形成能力,5μmol/L Triapine對Hep3B +p53DNA閤成的抑製率為40.89%,對細胞剋隆形成能力的抑製率達到68.16%,與Hep3B比較差異有統計學意義(P<0.05).Triapine作用後Hep3B+p53的Caspase-3能迅速激活,活性高峰提前齣現于6h.結論 p53在覈糖覈苷痠還原酶抑製劑化療藥物對肝癌細胞的GADD45誘導中具有重要作用,其作用機製可能是增彊轉錄調節因子的錶達水平.
목적 탐토3-안기필정-2-갑철류대축안기뇨(Triapine)대불동p53상태간암세포적생장억제급DNA손상유도기인45β(GADD45β)유도차이급기조공궤제.방법 전염p53전서렬건립Hep3B+p53;체외합성GADD45β근단6개계동자서렬군(-618 ~-314),구건형광소표체질립,전염간암세포주HepG2、Hep3B화Hep3B+p53;이실시형광정량취합매련반응(FQ-PCR)비교Triapine대GADD45β표체유도급기근단계동자활성영향차이;진일보비교대DNA합성화세포극륭형성능력억제차이;병통과반광안선천동안산특이성단백매(Caspase)-3적표체변화측정세포조망적발생화발전.결과 Triapine대Hep3B중GADD45유도병불명현,Hep3B+p53대Triapine민감성명현증가,2.5、5.0μmol/L제량하GADD45/감유철-3-린산탈경매(GAPDH)비치분별위0.0425화0.0704,병정현출제량-효응적정상관관계.형광소분석제시Triapine작용Hep3B+p53후적계동자핵인자(NF)-κB(-618/+6)화E2F-1(-470/+6)활성교Hep3B명현증강약1.3배화0.7배.Triapine능구경가명현지억제Hep3B +p53적DNA합성능력화세포극륭형성능력,5μmol/L Triapine대Hep3B +p53DNA합성적억제솔위40.89%,대세포극륭형성능력적억제솔체도68.16%,여Hep3B비교차이유통계학의의(P<0.05).Triapine작용후Hep3B+p53적Caspase-3능신속격활,활성고봉제전출현우6h.결론 p53재핵당핵감산환원매억제제화료약물대간암세포적GADD45유도중구유중요작용,기작용궤제가능시증강전록조절인자적표체수평.
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.