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为了研究c-myc基因反义寡核苷酸(ASODN)对HL-60细胞端粒酶活性的影响及其诱导凋亡作用,探讨HL-60细胞端粒酶活性与c-myc基因表达的关系,应用反义寡核苷酸封闭HL-60细胞c-myc基因的表达,RT-PCR方法检测该基因表达抑制情况,采用流式细胞术进行细胞凋亡检测及细胞周期分析,琼脂糖凝胶电泳观察凋亡细胞的DNA断裂情况,应用TRAP-ELISA法测定HL-60细胞端粒酶的活性。结果表明:反义硫代寡核苷酸作用于HL-60细胞72小时后,c-myc基因表达明显受抑;S期细胞百分数由55.6%降至30%,并出现早期凋亡峰(凋亡细胞比例占25.2%);琼脂糖凝胶电泳显示DNA凋亡梯形带;端粒酶活性检测发现反义寡核苷酸3,4,5μmol/L作用组OD450-690分别为1.952±0.14,1.805±0.40,1.616±0.41,与未作用组(OD450-690为2.648±0.42)比较,差异有显著性(P<0.05);反义寡核苷酸1和2μmol/L作用组及正义寡核苷酸5μmol/L作用组OD450-690分别为2.324±0.36,2.162±0.38,2.466±0.29,与未作用组比较,差异无显著性(P>0.05)。结论:c-myc基因反义寡核苷酸能够通过封闭c-myc基因的表达而诱导HL-60细胞凋亡,阻止细胞由G1期进入S期,并具有下调端粒酶活性作用。
To investigate the effect of c-myc antisense oligonucleotide (ASODN) on telomerase activity in HL-60 cells and its role in inducing apoptosis, we investigated the relationship between telomerase activity and c-myc gene expression in HL-60 cells The antisense oligonucleotide was used to block the expression of c-myc gene in HL-60 cells. The expression of c-myc gene was detected by RT-PCR. The apoptosis and cell cycle were analyzed by flow cytometry. Sepharose The DNA fragmentation of apoptotic cells was observed by electrophoresis. The telomerase activity of HL-60 cells was determined by TRAP-ELISA. The results showed that the expression of c-myc gene was significantly inhibited after antisense oligonucleotide treatment on HL-60 cells for 72 hours. The percentage of cells in S phase decreased from 55.6% to 30%, and the early apoptotic peak appeared Apoptotic cells accounted for 25.2%); DNA ladder electrophoresis showed DNA ladder; telomerase activity assay found that antisense oligonucleotides 3, 4, 5μmol / L effect group OD450-690 were 1.952 ± 0.14, 1.805 ± 0.40 and 1.616 ± 0.41, respectively, which was significantly different from that of the untreated group (OD450-690: 2.648 ± 0.42) (P <0.05). Antisense oligodeoxynucleotides 1 and 2 μmol / L group and sense oligo The OD450-690 of 5μmol / L group was 2.324 ± 0.36, 2.122 ± 0.38 and 2.466 ± 0.29, respectively. There was no significant difference between the untreated group and the untreated group (P> 0.05). CONCLUSION: Antisense c-myc antisense oligodeoxynucleotides induce the apoptosis of HL-60 cells by blocking the expression of c-myc gene and prevent the cells from entering the S phase from G1 phase and have the effect of down-regulating telomerase activity.