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目的:研究针对c-myc基因的小分子干扰RNA(siRNA)对HL-60细胞增殖及内源性c-myc基因表达的影响,探讨应用siRNA进行白血病基因治疗的可能性。方法:取倍增期HL-60细胞分空白对照组、阴性对照组、转染组3组,培养24～96h后收获细胞进行检测。采用MTT法检测细胞增殖状态,流式细胞仪进行细胞周期分析,逆转录PCR方法检测c-myc mRNA表达的变化,免疫细胞化学染色法检测C-myc蛋白表达的改变。结果:分组培养后,转染组细胞增殖较2个对照组受到抑制(P均<0.05),其抑制作用于转染后48、72h最明显(P均<0.05)。转染组S期细胞比值由空白对照组的(56.1±4.7)%降至(29.2±3.5)%,G0/G1期细胞由(36.8±3.3)%升高至(53.6±4.3)%,差异有统计学意义(P均<0.05)。c-myc siRNA对目的基因mRNA表达的抑制作用于24h最明显(P<0.05),96h抑制作用基本消失(P>0.05)。转染组细胞中C-myc蛋白表达与2个对照组相比降低(P均<0.05)。结论:针对c-myc基因的siRNA对HL-60细胞增殖及内源性c-myc基因表达有抑制作用,有望为白血病提供新的治疗靶位点。
Objective: To study the effect of small interfering RNA (siRNA) targeting c-myc gene on the proliferation of HL-60 cells and the expression of endogenous c-myc gene, and to explore the possibility of using siRNA for leukemia gene therapy. Methods: HL-60 cells in doubling phase were divided into blank control group, negative control group and transfected group. The cells were harvested 24-96 hours and harvested for detection. Cell proliferation was detected by MTT assay. Cell cycle analysis was performed by flow cytometry. The expression of c-myc mRNA was detected by RT-PCR. The expression of C-myc protein was detected by immunocytochemical staining. Results: The proliferation of the transfected cells was inhibited (P <0.05) and the inhibitory effect was most obvious at 48 and 72 hours after transfection (P <0.05). In the transfected group, the percentage of cells in the S phase decreased from (56.1 ± 4.7)% to (29.2 ± 3.5)% in the blank control group and from (36.8 ± 3.3)% to (53.6 ± 4.3)% in the G0 / There was statistical significance (all P <0.05). The inhibitory effect of c-myc siRNA on the mRNA expression of the target gene was the most obvious at 24h (P <0.05), but disappeared at 96h (P> 0.05). The expression of C-myc in transfected cells was lower than that in the two control groups (all P <0.05). CONCLUSIONS: The siRNA targeting c-myc gene has an inhibitory effect on the proliferation of HL-60 cells and the expression of endogenous c-myc gene, which may provide a new therapeutic target for leukemia.