反义寡聚脱氧核苷酸抗K562细胞的实验研究

Effect of Phosphorothiods Oligodeoxynucleotides on K562 Cells

针对慢性粒细胞白血病(CML)特征基因bcr/abl的嵌合位点和CML相关癌基因c-myb的第2外显子分别设计、合成的反义寡聚脱氧核苷酸asODN及其硫代磷酸化修饰物s-asODN分别或联合作用于慢性粒细胞白血病细胞株K562.结果表明各种asODN可显著抑制K562细胞的存活,最大抑制率为64.7%±3.2%,可显著抑制K562细胞的DNA合成,最大抑制率为85.8%±4.1%,可显著降低甚至几乎完全抑制bcr/abl的转录,可显著诱导K562发生凋亡.s-asODN的作用效果显著优于未加修饰的对应asODN.联合使用bcr/abl与c-myb的ASO比单独使用其中之一效果更佳.各种作用效果均表现出片断、时间相关性.这些说明,bcr/abl在CML发病机制中起双重作用,不仅刺激了CML细胞的过度增殖,而且抑制了细胞的凋亡.c-myb可能主要通过对bcr/abl的调控而与CML相关.asODN修饰物及多个癌基因的asODN联合使用可能是反义基因治疗的发展方向.

The respective sense and antisense oligodeoxynucleotides and their phosphorothiods, targeting at the bcr/abl, were the hallmark gene of Chronic Myelogenous Leukemia( CML) and c-myb correlates with CML. K562 cells, derived initially from a patient of CML blast crisis, were incubated with bcr/abl antisense oligodeoxynucleotides asODN or c-myb asODN or with both asODN in combinalion. All kinds of asODN could not only significantly inhibit K562 cells survival with the highest inhibitory rate of 64.7%±3.2%, but also dramatically inhibit DNA synthesis and the highest inhibitory rate was 85. 8 %±4.1%, decrease bcr/abl mRNA level almost completely and induce apoptosis significantly. The inhibition effect of antisense oligodeoxynucleotides phosphorothiodes s-asODN was stronger than that of unmodified asODN. The inhibition of the combination of bcr/abl and c-myb asODN was stronger than that of anyone alnoe. All the inhibitions exhibited in sequence-and time-dependent manner. We concluded that bcr/abl in pathogenesis of CML not only increased CML cells proliferation rate, but also decreased CML cells apoptosis rate. c-myb may participate in CML mainly by regulating bcr/abl. Trie results indicated that the modified asODN and multiple asODN targeting several oncogenes may advance to antisense gene therapy.