摘要
采用Oligofectamine转染靶向AKT的siRNA至人乳腺癌细胞系MCF-7,利用real-timePCR检测AKT的表达水平;MTT及流式细胞术分析转染后细胞的生物学特征变化;免疫荧光染色及Western印迹方法观察IA型PI3K/AKT通路主要成员的表达变化。Real-time PCR结果表明转染靶向AKT的siRNA组可以有效敲低AKT的表达水平;MTT结果显示AKT siRNA治疗组细胞增殖率显著降低;流式细胞术结果显示AKT siRNA转染组细胞在G_0/G_1期阻滞,凋亡比例明显高于空白对照组与空载体治疗组;免疫荧光和Western印迹结果均表明转染AKT siRNA组细胞AKT、pAKT、Ki67、Bcl-2几个重要癌蛋白的表达水平均有明显的下调。以上结果表明运用AKT siRNA转染人乳腺癌细胞系MCF-7细胞后,可抑制其肿瘤细胞的增殖并诱导凋亡,因此AKT可以作为人乳腺癌基因治疗的候选靶点。
Oligofectamine was used to transfect AKT siRNA to knock down the AKT expression level in MCF-7 human breast cancer cells. Real-time PCR was conducted to detect the expression of AKT mRNA. MTT and flow cytometry analysis were performed to detect the biological activity alternation after cells treated with AKT siRNA. Protein expression was evaluated by immunofluorescence staining and Western blot. The expression of AKT detected by RT-PCR was dramatically regressed in AKT siRNA-treated cells. Data from MTT assay indicated that growth was delayed for MCF-7 cells treated with AKT siRNA. Cell cycle was arrested in G0/G1 phase and the percentage of cell apoptosis increased significantly for the treated cells, which were determined by flow cytometry analysis. The result of immunofluorescence shown that the level of AKT, pAKT, Ki67, Bcl-2 were down-regulated which were also proved by Western blotting. Thus, AKT siRNA can effectively suppress the growth of MCF-7 cells and induce cell apoptosis, which suggests that AKT can be a candidate for gene therapy of human breast cancer.
出处
《细胞生物学杂志》
CSCD
2009年第6期837-842,共6页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30670802)
天津市应用基础与前沿计划重点项目(No.09JCZDJC19700)资助~~
