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目的:研究腺病毒介导14-3-3.σ(Ad-14-3-.3σ)对Akt过表达Rat1-Akt细胞成瘤性的作用,并探讨其作用是否通过负调控Akt而实现。方法:通过半体内和体内实验观察Ad-14-3-.3σ转染对Rat1-Akt细胞在裸鼠中成瘤性的影响;采用W estern b lotting方法检测转染14-3-.3σ基因后肿瘤组织内14-3-.3σ蛋白及其对Akt蛋白、Akt磷酸化活性和Akt磷酸化底物水平的影响。结果:无论体外用Ad-14-3-3σ处理Rat1-Akt细胞,还是体内经瘤内注射Ad-14-3-3σ,均可见14-3-3.σ可使荷瘤鼠肿瘤体积显著缩小(P<0.05),出现肿瘤的时间推迟,其中以长时间不间断给药疗效最好;转染14-3-.3σ基因治疗组的肿瘤组织中Akt蛋白、Akt-Thr308位点磷酸化活性及Akt磷酸化底物水平低于转染Ad-β-gal或PBS处理的对照组。结论:14-3-3σ可抑制Rat1-Akt细胞在裸鼠中的成瘤性,14-3-3σ通过负性调控Akt蛋白水平和磷酸化活性而抑瘤。
AIM: To investigate the effect of adenovirus-mediated 14-3-3.σ (Ad-14-3-.3σ) on the tumorigenicity of Akt-overexpressing Rat1-Akt cells and to explore whether its effect is mediated by negative regulation of Akt. METHODS: The effect of Ad-14-3-.3σ transfection on the tumorigenicity of Rat1-Akt cells in nude mice was observed by in vitro and in vivo experiments. The expression of 14-3-.3σ gene was detected by Western b lotting method 14-3-.3σ protein in tumor tissue and its effect on Akt protein, Akt phosphorylation activity and Akt phosphorylation substrate level. Results: Both in vivo and in vivo injection of Ad-14-3-3σ by either Ad-14-3-3σ treatment of Rat1-Akt cells showed that tumor volume of tumor-bearing mice was significantly reduced by 14-3-3σ (P <0.05). The tumor time was postponed, of which the curative effect was the best with uninterrupted administration for a long time. The Akt-Thr308 phosphorylation activity in the tumor tissues transfected with 14-3-.3σ gene therapy group And Akt phosphorylation substrate levels were lower than those transfected with Ad-β-gal or PBS. Conclusion: 14-3-3σ inhibits the tumorigenicity of Rat1-Akt cells in nude mice, and 14-3-3σ inhibits tumor cells by negatively regulating Akt protein level and phosphorylation activity.