Cyclin D1基因沉默对K562细胞增殖及凋亡的影响

[目的]利用RNA干扰（RNAi）技术观测其对白血病K562细胞cyelin D1基因的沉默效应及对细胞增殖、细胞周期及凋亡的影响。[方法]体外构建靶向cyclin D1基因的小发夹RNA（shRNA）表达质粒．通过壳聚糖介导转染K562细胞，Western blot分析检测转染前后cyclin D1蛋白表达变化；集落形成实验检测细胞增殖能力；流式细胞仪检测细胞周期分布及凋亡情况。[结果]构建靶向cyclinD1基因的shRNA表达质粒（pshRNA-419和pshRNA-575）经壳聚糖转染后，能显著抑制cyelin D1基因表达；抑制K562细胞增殖，影响其集落形成能力；细胞阻滞于G0／G1期，细胞凋亡明显。而m—pshRNA-790质粒并无上述生物学效应：[结论]cyclinD1基因表达下调可抑制K562细胞生长影响细胞周期分布并诱导细胞凋亡提示，因可能是白血病治疗的一个有效靶点。

Effect of Silencing Cyclin D1 Gene on Growth and Apoptosis of K562 Cells

[Purpose] To investigate the silencing effect of cyclin D1 gene in leukemia K562 cells by RNA interference technique and the influeuce on cell growth, cell cycle and apoptosis. [Methods] Plasmid vectors expressing small hairpin RNA （shRNA） targeting at cyclin D1 gene were constructed and transfected into K562 cells mediated by chitosan. Expression of Cyclin D1 protein before and after transfection was examined using Western blot analysis. Cellular proliferation was evaluated by soft agar colony formation assay. The cell cycle and apoptosis were determined by flow eytometry. [Results] Expression of cyclin D1 protein was markedly down-regulated, proliferation of K562 cells was inhibited, and capability of colony formation was suppressed after transfection with pshRNA419 and pshRNA-575 mediated by chitosan. K562 ceils were arrested at G0/G1 phase, and cell apoptosis was induced significantly. But no above biological effects was found in m-pshRNA-790 plasmid. [Conclusion ] Down-regulation of cyclin D1 may inhibit growth of K562 ceils, and induce cell apoptosis. It suggests that cyclin D1 gene might serve as an effective target for the treatment of leukemia.