中国医药生物技术
中國醫藥生物技術
중국의약생물기술
CHINESE MEDICINAL BIOTECHNOLOGY
2012年
5期
333-339
,共7页
王燕%李电东%邵荣光%王真
王燕%李電東%邵榮光%王真
왕연%리전동%소영광%왕진
肿瘤抑制蛋白质 p53%基因,报告%药物筛选试验,抗肿瘤%周期素依赖激酶抑制剂 p21
腫瘤抑製蛋白質 p53%基因,報告%藥物篩選試驗,抗腫瘤%週期素依賴激酶抑製劑 p21
종류억제단백질 p53%기인,보고%약물사선시험,항종류%주기소의뢰격매억제제 p21
Tumor suppressor protein p53%Genes, reporter%Drug screening assays, antitumor%Cyclin-dependent kinase inhibitor p21
目的构建含有 p53蛋白结合位点的 p21或 survivin 基因启动子区的荧光素酶报告基因重组质粒,并获得稳定转染的细胞模型用于抗肿瘤化合物筛选.
方法将含有 p53蛋白结合位点的 p21或 survivin 启动子区序列连接到 pGL4.17-basic 载体上,分别获得报告基因重组质粒 pGL4.17-p21和 pGL4.17-survivin;将两种重组质粒分别转染 A549细胞,经 G418筛选获得稳定转染细胞株 A549-p21和 A549-survivin;对该细胞模型进行条件优化,并将可调节 p53蛋白表达的多种抗肿瘤药物作用于稳定转染细胞,通过荧光素酶检测和 western blot 方法验证细胞模型用于药物筛选的可行性.
结果成功建立了分别含有 p21或 survivin 启动子报告基因的 A549-p21和A549-survivin 稳定细胞模型,多种抗肿瘤药物作用该细胞后能够通过调节 p53蛋白表达而影响报告基因荧光素酶活性.
结论构建的稳定转染细胞模型可以用于筛选以 p53为靶点的抗肿瘤化合物.
目的構建含有 p53蛋白結閤位點的 p21或 survivin 基因啟動子區的熒光素酶報告基因重組質粒,併穫得穩定轉染的細胞模型用于抗腫瘤化閤物篩選.
方法將含有 p53蛋白結閤位點的 p21或 survivin 啟動子區序列連接到 pGL4.17-basic 載體上,分彆穫得報告基因重組質粒 pGL4.17-p21和 pGL4.17-survivin;將兩種重組質粒分彆轉染 A549細胞,經 G418篩選穫得穩定轉染細胞株 A549-p21和 A549-survivin;對該細胞模型進行條件優化,併將可調節 p53蛋白錶達的多種抗腫瘤藥物作用于穩定轉染細胞,通過熒光素酶檢測和 western blot 方法驗證細胞模型用于藥物篩選的可行性.
結果成功建立瞭分彆含有 p21或 survivin 啟動子報告基因的 A549-p21和A549-survivin 穩定細胞模型,多種抗腫瘤藥物作用該細胞後能夠通過調節 p53蛋白錶達而影響報告基因熒光素酶活性.
結論構建的穩定轉染細胞模型可以用于篩選以 p53為靶點的抗腫瘤化閤物.
목적구건함유 p53단백결합위점적 p21혹 survivin 기인계동자구적형광소매보고기인중조질립,병획득은정전염적세포모형용우항종류화합물사선.
방법장함유 p53단백결합위점적 p21혹 survivin 계동자구서렬련접도 pGL4.17-basic 재체상,분별획득보고기인중조질립 pGL4.17-p21화 pGL4.17-survivin;장량충중조질립분별전염 A549세포,경 G418사선획득은정전염세포주 A549-p21화 A549-survivin;대해세포모형진행조건우화,병장가조절 p53단백표체적다충항종류약물작용우은정전염세포,통과형광소매검측화 western blot 방법험증세포모형용우약물사선적가행성.
결과성공건립료분별함유 p21혹 survivin 계동자보고기인적 A549-p21화A549-survivin 은정세포모형,다충항종류약물작용해세포후능구통과조절 p53단백표체이영향보고기인형광소매활성.
결론구건적은정전염세포모형가이용우사선이 p53위파점적항종류화합물.
Objective To construct a luciferase reporter gene recombinant plasmid containing human p21 or survivin gene promoter region with p53 binding site, and to obtain cell lines stably transfected with the recombinant plasmid.
@@@@Methods DNA fragments containing p21 or survivin promoter region were inserted to pGL4.17-basic plasmid to construct pGL4.17-p21 and pGL4.17-survivin reporter plasmid, respectively. A549 cells were stably tranfected with the recombinant plasmids to obtain the cellular screening models. The screening models were then optimized and verified by some known p53-targeted anti-cancer drugs with luciferase assay and western blot.
@@@@Results Two cellular models stably transfected with pGL4.17-p21 or pGL4.17-survivin reporter plasmid were successfully constructed and named as A549-p21 and A549-survivin, respectively. Several anti-cancer drugs known as regulators of p53 expression were used to verify the validity of the screening models.
@@@@Conclusion The constructed cellular models can be used for identification of lead compounds that may regulate p53 expression and have the anti-cancer potential.
