Survivin联用bcl-2反义寡核苷酸对白血病细胞凋亡的诱导作用

Combination of survivin and bcl-2 antisense oligodeoxynucleotides in the induction of K562 cell apoptosis

目的:探讨survivin、bcl-2反义寡核苷酸(ASODN)联用对白血病细胞凋亡的诱导作用。方法:设计并合成靶向survivin ASODN和bcl-2的ASODN,应用脂质体Lipofectamine TM2000作为载体,转染寡核苷酸入白血病K562细胞。实验分为空白对照组、脂质体空转染组、无关序列寡核苷酸组、survivin ASODN组、bcl-2ASODN组和survivin、bcl-2ASODN半量联用组。转染48h后采用四甲基偶氮唑蓝(MTT)法检测细胞增殖抑制率,流式细胞仪检测细胞凋亡率,RT-PCR半定量检测K562细胞survivin mRNA表达。结果:survivin ASODN和bcl-2ASODN对K562细胞均有一定的增殖抑制作用,抑制率分别为48.78%、40.24%。而survivin、bcl-2ASODN半量联用组的细胞增殖抑制率为60.98%,明显高于单用组(P<0.01)。与非ASODN组相比,survivin ASODN和bcl-2ASODN均可诱导K562细胞凋亡,凋亡率分别为(13.36±4.03)%、(10.40±1.71)%,两者联用凋亡率提高到(26.14±4.39)%(P<0.01)。Survivin、bcl-2ASODN半量联用组survivin mRNA水平明显低于survivin ASODN组和bcl-2ASODN组。结论:survivin、bcl-2ASODN联用可协同抑制K562细胞的增殖,增强凋亡诱导作用,可为白血病基因治疗提供一项新策略。

Objective To explore the synergistic effect of survivin and bcl-2 antisense oligodeoxynucleotide （ASODN） on the induction of K562 cell apoptosis. Methods The ASODNs were transfected into K562 cells by the vector Lipofectamine^TM2000. There were six groups in this experiment：control group, lipo group, NODN group, survivin ASODN group, bcl-2 ASODN group, and combination group. MTT assay was used to determine cell proliferation. The rate of cell apoptosis was detected by FCM. The expression of survivin mRNA was evaluated with semi-quantitative RT-PCR. Results The inhibitory rate of K562 cell was significant higher in the combination group than in survivin ASODN or bcl- 2 ASODN group （60.98% vs, 48.78% or 60.98% vs. 40.24%, P 〈 0.01 ）. Either survivin ASODN or bcl-2 ASODN could induce K562 cell apoptosis with a apoptotic rate of （13.36 ± 4.03）% or （10.40 ± 1.71 ）% and the rate elevated to （26.14 ± 4.39）% for the combination of survivin ASODN and bcl-2 ASODN （P 〈 0.01）. The level of survivin mRNA in the combination group was markedly lower than that in survivin ASODN or bcl-2 ASODN group. Conclusion The combination of survivin and bcl-2 ASODN synergistically inhibits the proliferation of K562 cells and increases cell apoptosis, providing a new gene therapy for leukemia.