存活素反义寡核苷酸对甲状腺癌细胞生长的抑制作用

The growth-inhibiting effect of survivin antisense oligonucleotide on thyroid carcinoma cells

目的探讨利用凋亡抑制基因存活素反义寡核苷酸(ASODN)诱导甲状腺癌细胞凋亡的效应。方法设计合成特异性靶向存活素的ASODN,以不同浓度和时间对人甲状腺髓样癌细胞进行转染,并设空白、正义(SODN)对照组进行比较。采用RT-PCR、Western印迹、免疫细胞化学技术检测各组细胞中存活素mRNA、蛋白表达水平,DNA裂解分析、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。结果转染ASODN各组细胞存活素mRNA和蛋白表达均较空白对照组、SODN组明显减弱;细胞凋亡率显著增加,细胞生长相应受抑,上述效应在ASODN转染24h最为明显,并呈浓度依赖性;而各时段SODN组与空白对照组间各项指标差异均无统计学意义。结论ASODN能够特异性地下调甲状腺癌细胞中存活素基因表达,进而诱导肿瘤细胞凋亡,并抑制其增殖。

Objective To investigate the inductive effect of survivin （an inhibitor of apoptosis gene） antisense oligonucleotide （ASODN） on apoptosis of medullar thyroid carcinoma cells. Methods Targeted ASODN of survivin was designed and synthesized and then transfected into human medullary thyroid carcinoma cell lines at different concentration and time; and simultaneously normal control and positive control （sense oligonucleotides, SODN group） were set for comparison. RT-PCR, Western blotting and immunecytechemical technique were used to detect the expressions of survivin mRNA and protein in each group; DNA ladder, acridine orange / ethidium bromide （AO/EB） staining and flow cytometer （FCM） were used to detect cell apoptosis and morphological change in each group; MTT assay was used to detect cell growth suppression. Results Compared with normal control and positive control, the expressions of survivin mRNA and protein in cells of each transfection ASODN group were obviously decreased, apoptosis rate was significantly increased, and cell growth was inhibited correspondingly. The effects mentioned above were in a concentration-dependent manner, and the most obvious effect was shown 24 hours after transfection with ASODN; there was no obvious difference between normal control and positive control at different times. Conclusion ASODN can down-regulate the expression of survivin gene in thyroid carcinoma specifically, induce tumor cell apoptosis and suppress cell proliferation.