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目的探讨核仁磷酸蛋白(nucleophosmin,NPM)在人乳腺癌耐药形成中的作用机制。方法采用低浓度持续诱导法建立甲氨蝶呤耐药的人乳腺癌细胞株(MCF-7/MTX);利用四甲基偶氮唑蓝(MTT)法测定细胞活性,绘制细胞生长曲线并计算倍增时间;倒置显微镜及透射电镜观察细胞形态;应用Western-blot和Real-time PCR测定NPM及耐药因子在细胞中的表达,运用siRNA干扰技术降低MCF-7/MTX细胞中NPM的表达,探究细胞的耐药机制。结果成功建立甲氨蝶呤耐药的人乳腺癌细胞株MCF-7/MTX,耐药倍数为64倍;耐药细胞生长偏慢呈梭形,并且内部结构发生变化;MCF-7/MTX细胞对多种化疗药物都具有交叉耐药性;NPM及多药耐药相关因子P-gp、MRP1、BCRP在耐药细胞株中的表达均上调;高表达的NPM进一步激活PI3K/Akt信号通路并抑制下游的凋亡因子;干扰降低NPM表达后,MCF-7/MTX细胞的耐药性明显降低,PI3K/Akt通路受到抑制促进下游的细胞凋亡。结论 NPM高表达在人乳腺癌耐药形成中起到重要的作用,其有望成为临床上治疗乳腺癌耐药的新的分子靶标。
Objective To investigate the mechanism of nucleophosmin (NPM) in the development of human breast cancer drug resistance. Methods Methotrexate-resistant human breast cancer cell line (MCF-7 / MTX) was established by continuous induction with low concentration. The cell viability was measured by MTT assay and the cell growth curve was calculated Doubling time; inverted microscope and transmission electron microscope observation of cell morphology; application of Western-blot and Real-time PCR determination of NPM and resistance factor in cells, using siRNA interference technology to reduce the expression of NPM in MCF-7 / MTX cells explore Cell resistance mechanism. Results Methotrexate-resistant human breast cancer cell line MCF-7 / MTX was successfully established, with a multiple of 64 fold. The growth of drug-resistant cells was spindle-shaped and the internal structure changed. MCF-7 / MTX cells The expression of P-gp, MRP1 and BCRP in drug-resistant cell lines were all up-regulated by NPM and the high expression of NPM further activated the PI3K / Akt signaling pathway Inhibition of downstream apoptosis factors; Interference reduced NPM expression, MCF-7 / MTX cell resistance was significantly reduced, PI3K / Akt pathway was inhibited to promote downstream apoptosis. Conclusion NPM overexpression plays an important role in the development of human breast cancer drug resistance. It is expected to become a new molecular target for clinical treatment of breast cancer drug resistance.