Bcr/abl融合基因的小干扰RNA对K562细胞增殖和凋亡的影响

目的 观察特异性bcr/abl融合基因的siRNA对慢性粒细胞白血病K562细胞增殖和凋亡的影响。方法　设计并化学合成bcr/abl融合基因融合位点b3:a2 21个核苷酸 siRNA作用于K562细胞，用WST-8法检测细胞增殖抑制率；RT-PCR 检测 bcr/abl mRNA表达水平；PI单染流式细胞仪检测细胞周期；Annexin V-PI双染测定细胞凋亡比例；Hochest33258 染色荧光显微镜观察细胞凋亡的形态学变化。结果 ①Bcr/abl siRNA作用K562细胞24h,48h,72h后，明显抑制K562细胞增殖，各浓度组间的增殖抑制率无明显差异(p>0.05)；②Bcr/abl siRNA能显着下调bcr/abl mRNA水平，各浓度组间差异无显著性(p>0.05)；③Bcr/abl siRNA作用组细胞周期阻滞于G1期；④Bcr/abl siRNA作用后细胞出现明显的早期凋亡群，各浓度组间的早期凋亡率差异无统计学意义(p>0.05)，细胞出现核固缩、核边集、凋亡小体等改变。结论 　特异性bcr/abl siRNA可显着抑制K562细胞bcr/abl融合基因的表达，抑制细胞的增殖，诱导凋亡，但其作用未显示明显的剂量依赖性。

Effect of bcr/abl fusion gene siRNA on proliferation and apoptosis of K562 cells

Abstract: Objective 　To investigate the effect of bcr/abl fusion gene small interfering RNA (siRNA) on proliferation and apoptosis of chronic myelogenous leukemia (CML) K562 cells. Methods　A 21nt siRNA targeting b3: a2 mRNA of bcr/abl fusion gene was designed, synthesized, and transfected into K562 cells.The proliferation inhibition rate was detected by WST-8 method; the expression of bcr/abl mRNA of K562 cells was detected by RT-PCR; the cell cycle was used to observed by Phosphatidylinositol (PI) staining; the apoptosis rate of; was detected by Annexin V-fluorescencein isothiocyanate/phosphatidylinositol (PI) double staining; the morphology of apoptosis was observed by Hochest33258 staining. Results　 (1) bcr/abl siRNA significantly inhibited K562 cells proliferation after 24h, 48h, 72h transfection. (2) bcr/abl siRNA significantly down-regulated mRNA expression level There was no significant difference between among different concentration bcr/abl siRNA groups (P>0.05). (3) Cell cycle of K562 cells was blocked in G1 phase after bcr/abl siRNA transfection.(4) Early apoptotic cells groups appeared after bcr/abl siRNA transfection.The early apoptotic rates were not significantly different among different concentration bcr/abl siRNA groups (P>0.05). Cells presented karyopyknoses, nuclear-set, apoptotic bodies. Conclusions　The specific bcr/abl fusion gene siRNA significantly inhibits the expression of bcr/abl fusion gene, inhibits K562 cells proliferation and induces its apoptosis , but the efficacy was not significant dose-dependent