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目的 :研究联合应用bcr abl融合基因硫代磷酸反义寡核苷酸 (Aspo)及c myb基因Aspo对K5 6 2细胞作用效果。 方法 :Aspo与K5 6 2细胞共培养后 ,台盼蓝拒染法计死活细胞数 ;甲基纤维素半固体培养法培养CFU K5 6 2 ;流式细胞仪测定细胞P2 10蛋白表达及细胞凋亡率 ;RT PCR半定量检测细胞bcr ablmRNA ;电镜观察凋亡细胞形态学改变。结果 :倒置显微镜下观察到联合应用两种Aspo时 ,浓度各为 5 μmol/L组K5 6 2细胞仍呈克隆状生长 ,作用 2 4h细胞P2 10蛋白表达明显受抑制 ,表达率 <2 % ;作用 12 0h细胞生长抑制率为 6 1.7% ,P2 10恢复为 2 5 .7% ,凋亡率为 2 2 .5 %。两种浓度各为 10 μmol/L组K5 6 2细胞生长疏散 ,作用 12 0h细胞生长抑制率达 92 .2 % ,P2 10仍未恢复 ,凋亡率为 6 4.3% ,电镜下见细胞呈典型凋亡形态学改变。当两种Aspo浓度各为 5 μmol/L及 10 μmol/L作用 48h时 ,K5 6 2细胞bcr ablmRNA较空白组下降 6 9.2 %～85 3%。结论 :联合应用bcr abl基因Aspo及c myb基因Aspo对K5 6 2细胞的抑制作用具有一定的协同性 ,可增强其抗白血病效果。
Objective: To study the effect of combination of bcr abl fusion gene phosphorothioate antisense oligonucleotide (Aspo) and c myb gene Aspo on K562 cells. Methods: After co-cultured with Aspo and K5 6 2 cells, the number of viable cells was counted by trypan blue exclusion method. CFU K5 6 2 was cultured with methylcellulose semi-solid culture method. The expression of P2 10 protein and apoptosis were detected by flow cytometry The rate of apoptosis was detected by semi-quantitative RT-PCR. The morphological changes of apoptotic cells were observed under electron microscope. Results: Under the inverted microscope, the K562 cells with 5 μmol / L K562 were still clonally cultured. The expression of P2 10 protein was significantly inhibited at 2 hours and the expression rate was less than 2%. The cell growth inhibition rate at 12 0h was 6 1.7%, P2 10 was 25.7%, and the apoptosis rate was 22.5%. The growth of K5 6 2 cells in two concentrations of 10 μmol / L was evacuated, the growth inhibition rate reached 92.2% at 12 h, P2 10 was still not restored, the apoptosis rate was 6 4.3% Morphological changes of apoptosis. When the concentration of Aspo was 5 μmol / L and 10 μmol / L for 48 h, the bcr abl mRNA in K562 cells decreased by 62.2% ~ 85.3% compared with the blank group. CONCLUSION: The combination of asc in bcr abl gene and Aspo in c myb gene has a synergistic effect on the inhibition of K562 cells, which can enhance its anti-leukemia effect.