【摘要】 目的:利用Tet-on基因表达调控系统建立由强力霉素(Dox)诱导TAp63γ基因表达的EC9706细胞系,为进一步研究TAp63γ基因的功能奠定基础。方法:将调控质粒pTet-on转入EC9706细胞,经G418筛选后的细胞再次转染反应质粒pTRE-TAp63γ-HA,再用潮霉素筛选出阳性细胞克隆,RT-PCR法选择对Dox敏感的细胞克隆。最后用不同浓度Dox诱导,Westernblot法确定Dox的最佳诱导浓度。采用此浓度的Dox分别诱导EC9706、EC9706/Tet/pTRE、EC9706/Tet/pTRE-TAp63γ3组细胞,细胞增殖分析试剂盒(CCK-8试剂盒)检测细胞生长抑制率。结果:Dox浓度为3mg/L时可诱导TAp63γ基因高表达,EC9706/Tet/pTRE-TAp63γ细胞的生长抑制率均高于EC9706和EC9706/Tet/pTRE细胞(P<0.05或0.01)。结论:成功构建了Tet-on调控TAp63γ基因表达的EC9706细胞系,为进一步研究食管鳞癌细胞内P63蛋白的生物学行为提供了一个理想的研究平台。TAp63γ基因表达可使EC9706细胞增殖受到抑制。更多还原

【Abstract】 Aim: To establish the functional expression of TAp63γ with doxycycline (Dox) induced Tet-on regulating system in human esophageal squamous carcinoma cell EC9706, and provide an ideal experimental platform for further studies of TAp63γ.Methods:pTet-on regulating plasmid was transfected into EC9706,and stable expression of Tet-on was established in EC9706 through G418 selection. Then the response plasmid of recombinant pTRE-TAp63γ-HA was steadily transfected into positive EC9706/Tet-on cells with hygromycin screen. Dox was used to induce the expression of TAp63γ and a cell clone sensitive to Dox was selected through RT-PCR assay. The best-induced concentration was determined with different concentration of Dox induction by Western blot assay. The percentages of growth inhibition were detected by CCK-8 assay after TAp63γ gene up-regulated expression with Dox induction.Results: High TAp63γ expression was induced by Dox with the concentration of 3 mg/L. The percentages of growth inhibition of EC9706/Tet/pTRE-TAp63γ cells were significantly higher than those of EC9706 cell and EC9706/Tet/pTRE cell (P<0.05 or 0.01). Conclusion: Functional expression of TAp63γ under Dox induced Tet-on regulation system have been successfully established in EC9706, which provides an ideal experimental platform for the further study of TAp63γ in esophageal squamous carcinoma cell. The growth of EC9706 is inhibited by expression of TAp63γ.更多还原