Survivin shRNA促进HL-60细胞凋亡及其对阿糖胞苷敏感性的研究

目的研究RNA干扰Survivin基因表达对HL-60细胞凋亡及其对阿糖胞苷敏感性的影响。方法采用PCR扩增以及定向克隆技术重组构建shRNA的表达载体pshRNA-Survivin,使用脂质体介导转染至HL-60细胞,分为6组：①空白实验组;②阿糖胞苷（Ara-C）组;③阳性质粒组;④阴性质粒组;⑤阳性质粒＋Ara-C组;⑥阴性质粒＋Ara-C组。采用RT-PCR技术检测HL-60细胞内Survivin及其mRNA表达,运用流式细胞仪进行细胞凋亡率改变的检测。结果与空白实验组比较,阳性质粒组的mRNA表达抑制率为57.7%,表达下降,差异有统计学意义（P〈0.05）;阳性质粒组细胞凋亡率为5.87%,阳性质粒＋Ara-C组细胞对Ara-C敏感性明显提高,细胞凋亡率为12.4%,差异有统计学意义（P〈0.05）。结论 Survivin siRNA能够高效特异的抑制HL-60细胞内Survivin基因的表达,诱导细胞凋亡,并提高了HL-60细胞对Ara-C的敏感性,这有助于白血病基因治疗的进一步发展。

Investigation of Survivin shRNA on the apoptosis and sensitivity to ARa- C of HL-60 cells

Objective To Investigate the effect of Survivin siRNA on the apoptosis and sensitivity to ARa-C of HL-60 cell. Methods PCR and cloned recombinant were used to construct siRNA expression vectors pshRNA-Survivin, then the expression vectors were transfected into HL-60 cell by Lipofectamine. All the cells were divided into 6 groups： （1）blank group; （2）Ara-C group; （3）positive plasmid group; （4）negative plasmid group; （5）positive plasmid＋Ara-C group; （6）negative plasmid Ara-C group. RT-PCR technique was used to detect the expression of Survivin mRNA and FCM was used to detect the apoptosis. Results mRNA express inhibition rate in positive plasmid group （57.7%） was lower than that in blank group, the difference was statistically significant （P 〈 0.05）; apoptosis rate in positive plasmid group was 5.87%, the expression was found declined, the susceptibility to Ara-C in positive plasmid＋Ara-C group was found improved, its apoptosis rate was 12.4%, the difference was statistically significant （P 〈 0.05）. Conclusion Survivin siRNA can inhibit the expression of Survivin mRNA of HL-60, induce apoptosis of HL-60 cell and increase the sensitivity of the HL-60 cell to Ara-C. It is helpful to the furtber development of leukemia gene therapy.