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目的探讨Livin反义寡核苷酸(ASODN)对白血病细胞K562增殖、凋亡和耐药的影响。方法设计合成特异性的Livin硫代磷酸ASODN及其对照错义寡核苷酸(MSODN),脂质体(Lip)介导转染K562细胞,实验分为Lip-ASODN组、Lip-MSODN组、Lip对照组和细胞对照组4组,采用反转录-PCR法检测Livin mRNA的表达;四甲基偶氮唑蓝法与膜联蛋白V异硫氰酸荧光素法检测Livin ASODN与K562细胞增殖、凋亡及与高三尖杉酯碱(HHT)耐药的关系。结果 Livin ASODN可显著抑制K562细胞增殖(P<0.01),且作用具有时间和浓度依赖性。终浓度为600 nmol.L-1的Lip-ASODN能明显降低Livin mRNA表达,细胞凋亡率达(36.89±1.08)%(P<0.01),而Lip-MSODN组、Lip对照组与细胞对照组之间差异均无统计学意义(Pa>0.05)。Livin ASODN可增加K562细胞对HHT的耐药性,半数抑制质量浓度为(0.37±0.02)mg.L-1;与HHT及小剂量阿糖胞苷联合应用可显著抑制细胞增殖(P<0.01)。结论 Livin ASODN可下调Livin基因表达,有效抑制K562细胞的增殖,增加K562细胞凋亡及其对HHT的敏感性。
Objective To investigate the effect of Livin antisense oligonucleotide (ASODN) on the proliferation, apoptosis and drug resistance of leukemia K562 cells. Methods Specific synthetic Livin phosphorothioate ASODN and its control missense oligodeoxynucleotide (MSODN) were designed and synthesized. Lipofectamine 2000 was transfected into K562 cells. The experiment was divided into Lip-ASODN group, Lip-MSODN group, Lip control group and cell control group, the expression of Livin mRNA was detected by reverse transcription-PCR; the proliferation of Livin ASODN and K562 cells was detected by MTT assay and annexin V fluorescein isothiocyanate assay , Apoptosis and its relationship with HHT resistance. Results Livin ASODN could significantly inhibit the proliferation of K562 cells (P <0.01), and its effect was time-and dose-dependent. Lip-ASODN at a final concentration of 600 nmol.L-1 could significantly decrease the expression of Livin mRNA and the rate of apoptosis was (36.89 ± 1.08)% (P <0.01), while Lip-MSODN group, Lip control group and cell control group The difference was not statistically significant (Pa> 0.05). Livin ASODN could increase the drug resistance of H5 in K562 cells. The half-inhibitory concentration of Livin ASODN was (0.37 ± 0.02) mg.L-1. HHT and low dose of cytarabine could inhibit cell proliferation significantly (P <0.01) . Conclusion Livin ASODN can down-regulate the expression of Livin gene, effectively inhibit the proliferation of K562 cells and increase the apoptosis of K562 cells and its sensitivity to HHT.