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为研究c myc硫代反义寡脱氧核苷酸对白血病细胞系HL 60诱导细胞凋亡的作用 ,将人工合成的长度分别为 18mer和 15mer的硫代反义寡脱氧核苷酸 (AspoⅠ和AspoⅡ )转染HL 60细胞 ,用流式细胞术检测c Myc蛋白及DNA倍体分析 ,RT PCR检测c mycmRNA水平 ,电镜观察凋亡细胞的超微结构 ,琼脂糖凝胶电泳观察细胞DNA降解片段。研究结果发现 ,经AspoⅠ 5 μmol L和 10 μmol L作用后c Myc蛋白分别降低 2 3.8%和 4 5 .4 % ,经AspoⅡ 10 μmol L作用后下降38.4 % ,RT PCR显示AspoⅠ和AspoⅡ组c mycmRNA表达明显减少。DNA倍体分析AspoⅠ 10μmol L作用 4 8和 72小时细胞凋亡率分别为 2 3.97%和 5 2 .6% ;AspoⅡ 10 μmol L作用 4 8和 72小时后细胞凋亡率分别为 2 8.8%和 4 5 .19% ;而空白对照和正义寡脱氧核苷酸组均未出现凋亡细胞。电镜观察两个反义寡脱氧核苷酸 10 μmol L作用后均出现细胞固缩 ,染色质边集 ,胞浆浓缩等凋亡细胞的改变。反义寡脱氧核苷酸 10 μmol L作用 4 8小时后均出现典型的DNA降解片段。本研究结果说明 ,c myc反义寡脱氧核苷酸是高特异性的基因药物 ,对HL 60细胞抑制c mycmRNA的表达 ,减少c Myc蛋白合成 ,并诱导HL 60细胞凋亡
To investigate the effect of c myc thio-antisense oligodeoxynucleotide on apoptosis of leukemic cell line HL 60, artificially synthesized thiol antisense oligodeoxynucleotides of 18 mer and 15 mer in length (Aspo I and Aspo II HL 60 cells were transfected, c Myc protein and DNA ploidy analysis were detected by flow cytometry, c myc mRNA levels were detected by RT PCR, ultrastructure of apoptotic cells was observed by electron microscopy, and DNA fragmentation was observed by agarose gel electrophoresis. The results showed that the protein levels of c Myc were decreased by 23.8% and 45.4%, respectively, after treatment with AspoI 5 μmol L and 10 μmol L, and decreased by 38.4 % after being treated with Aspo II 10 μmol L. RTPCR showed c myc mRNA in Aspo I and Aspo II groups. The expression was significantly reduced. DNA ploidy analysis showed that apoptosis rate of AspoI at 10 μmol L for 48 and 72 hours was 23.97% and 52.6%, respectively; AspoII at 10 μmol L for 48 and 72 hours, the apoptotic rate was 28.8%. 4 519.9% did not show apoptotic cells in the control and sense oligodeoxynucleotide groups. Electron microscopy observations of the effect of 10 μmol L of two antisense oligodeoxynucleotides all showed changes in apoptotic cells such as cell shrinkage, chromatin condensation and cytoplasm condensation. Antisense oligodeoxynucleotides (10 μmol L) showed typical DNA degradation fragments after 48 hours. The results of this study indicate that c myc antisense oligodeoxynucleotide is a highly specific gene drug that inhibits the expression of c myc mRNA in HL 60 cells, reduces c Myc protein synthesis, and induces apoptosis in HL 60 cells.