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目的利用 RNA 干扰(RNAi)技术体外观测其对 K562细胞 cyclin 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率的影响。结果构建靶向 cyclin D1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 cyclin D1基因表达;抑制 K562细胞增殖,影响其集落形成能力;影响 K562细胞周期分布,诱导细胞凋亡。而设计一碱基突变的序列所构建的质粒并无上述生物学效应。结论 cyclin D1基因表达下调可抑制 K562细胞生长,并诱导细胞凋亡。提示 cyclin D1基因可能是白血病治疗的一个有效靶点的。
Objective To observe the effects of silencing cyclin D1 gene on the proliferation and cell cycle and apoptosis of K562 cells by RNA interference (RNAi) technique. Methods K562 cells were transfected with chitosan and the expression of Cyclin D1 was detected by Western blot. The colony formation assay was used to detect the proliferation of K562 cells. The cell cycle distribution and apoptosis rate were detected by flow cytometry. Results After transfected with chitosan, the shRNA expression plasmids (pshRNA-419 and pshRNA-575) targeting cyclin D1 gene could significantly inhibit the expression of cyclin D1 gene, inhibit the proliferation of K562 cells and affect the colony formation ability of K562 cells, Periodic distribution induces apoptosis. The design of a single nucleotide mutation in the constructed plasmid did not have the above biological effects. Conclusion The down-regulation of cyclin D1 gene can inhibit the growth of K562 cells and induce apoptosis. This suggests that cyclin D1 gene may be an effective target for the treatment of leukemia.