Survivin反义寡核苷酸对骨肉瘤细胞凋亡的诱导效应

Inducible effect of antisurvivin oligonucleotide on apoptosis in osteosarcoma

目的:利用骨肉瘤OS-732细胞,探讨survivin反义寡核苷酸(ASODN)对survivin基因表达的影响及其诱导肿瘤细胞凋亡的效应。方法:设计合成特异性靶向survivin的ASODN,以不同浓度和时间对OS-732细胞进行转染,并设空白、正义(SODN)对照组进行比较。采用RT-PCR、Westernblotting、免疫细胞化学技术检测各组OS-732细胞中survivinmRNA、蛋白表达状态,吖啶橙/溴化乙锭(AO/EB)染色法、流式细胞仪检测细胞凋亡水平和形态变化,MTT法检测细胞生长抑制情况,激酶活性测定法检测细胞中caspase-3活性程度。结果:转染ASODN各组OS-732细胞survivinmRNA和蛋白表达均明显弱于空白对照组、SODN组;细胞凋亡率(AI)显著增加,细胞生长相应受抑,caspase-3活性显著提高;上述效应在ASODN各组存在一定的时间浓度依赖性;而SODN各组与空白对照组间各项指标均无明显差异。结论:ASODN能够特异性下调OS-732细胞中survivin基因表达,激活凋亡效应蛋白酶caspase-3,在诱导肿瘤细胞凋亡、抑制增殖中发挥重要作用。

AIM： Osteesarcoma OS- 732 cell line was used to investigate the effect of antisense oligonucleotide （ASODN） on survivin expression and its inducible effect on tumor cells apoptesis. METHODS： ASODN of specific target survivin was designed, synthesized and then transfer to OS - 732 cell line with different concentrations and time points. At the same time blank control group, sense oligonucleotide （SOND） group were set up for comparison. Reverse tanscriptionase- polymerase chain reaction （ RT- PCR）, Western blotting and immtmohistochemistry were used to detect the expressions of survivin mRNA and protein in each OS - 732 cell line group. Acridine orange/ethidium bromide （AO/EB） staining and flow cytometry were used to detect the apoptosis level and morphologic change. Mononuclear cell direct cytotoxicity assay （MTT） was used to estimate cell growth suppression. Kinase activity assay method was used to estimate the activity of caspase- 3 in the cells. RESULTS： Compared with control group and SOND group, in ASODN groups, the expression of survivin mRNA and protein were obviously weaken, apoptosis rate and easpase - 3 activity apparently increased, cells growth was inhibited. In each ASODN group, the effect above - mentioned has time - and concentration- dependent manner. There was no obviously difference of each index in each SODN and blank control groups. CONCLUSION： ASODN down- regulated the expression of survivin gene in OS- 732.cell line specifically, and activated apoptosis effectively. It plays an important role in inducing tumor apoptosis and suppressing cell proliferation.