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目的 观察caspase 3mRNA反义寡核苷酸 (ASODN)对HL 6 0细胞凋亡的抑制作用 ,筛选有效ASODN。 方法 用脂质体介导法将针对caspase 3mRNA不同序列的 4条ASODN导入HL 6 0细胞中 ,γ 射线照射。应用电泳法检测DNA梯状条带 ;Hoechst332 5 8 碘化丙啶染色 ,荧光显微镜分析凋亡细胞百分率 ;流式细胞术进行细胞凋亡定量。 结果 以caspase 3mRNA 5′非编码区 (- 6 2至 - 4 6位 )与编码起始区 (- 1至 16位 )ASODN转染 ,当转染终浓度≥ 3μmol L时 ,DNA电泳梯状条带消失 ,流式细胞术亦未见明显的亚二倍体峰 ;荧光染色分析 ,凋亡细胞百分率比未转染对照组和错配寡核苷酸对照组显著降低 (P <0 0 1) ,且随转染终浓度的增加 ,凋亡抑制率显著增加。另外 ,5′非编码区ASODN的抑制作用显著强于编码起始区ASODN(P <0 0 5 )。 结论 caspase 3mRNAASODN可抑制γ 射线诱导的HL 6 0细胞凋亡 ,其作用有序列特异性及剂量依赖性
Objective To observe the inhibitory effect of caspase 3 mRNA antisense oligonucleotide (ASODN) on the apoptosis of HL-60 cells and screen the effective ASODN. Methods Four ASODNs targeting different sequences of caspase 3 mRNA were introduced into HL-60 cells by liposome-mediated method and irradiated with γ-rays. DNA ladder bands were detected by electrophoresis; Hoechst33258 staining with propidium iodide, the percentage of apoptotic cells were analyzed by fluorescence microscopy; and apoptosis was quantified by flow cytometry. Results ASODN was transcribed in the 5 ’untranslated region (-62 to - 46) of the caspase 3 mRNA together with ASODN encoding the initiation region (-1 to 16 positions). When the final concentration of transfection was ≥ 3 μmol L, There was no obvious sub-diploid peak by flow cytometry. Fluorescence staining showed that the percentage of apoptotic cells was significantly lower than that of untransfected control group and mismatched oligonucleotide control group (P <0.01) , And with the increase of the final concentration of transfection, the apoptosis inhibition rate increased significantly. In addition, the inhibitory effect of ASODN in 5 ’non-coding region was significantly stronger than ASODN in coding initiation region (P <0 05). Conclusion Caspase 3 mRNA ASODN can inhibit the apoptosis of HL-60 cells induced by γ-rays, and its effect is sequence-specific and dose-dependent